Patent application title: USE OF VALPROIC ACID FOR ENHANCING PRODUCTION OF RECOMBINANT PROTEINS IN MAMMALIAN CELLS

Inventors: Markus Hildinger (Pforzheim, DE) Gaurav Backliwal (Lausanne, CH) Florian Wurm (Lausanne, CH)
Assignees: EXCELLGENE SA
IPC8 Class: AC12P2100FI
USPC Class: 435 703
Class name: Animal tissue cell culture
Publication date: 01/22/2009
Patent application number: 20090023186

Abstract:

Culturing cells for the commercial production of proteins for diagnosis and therapy is a costly and time consuming process. The equipment required is expensive, and production cost are high. In order to provide commercially viable processes it is desirable to use cell lines which produce large quantities of product with each production run. However, most cells do not produce large quantities of desired product per se either because they do not produce a large quantity of product per unit of time (specific productivity) or because they do not survive long enough in the culture medium (time). Here, we identified that addition of a valproic acid compound to the culture medium increases overall (batch) yield and titer. More importantly, compared to the widely used sodium butyrate, batch yields using a valproic acid compound as a medium additive are significantly higher.

Claims:

1. A process for the production of a protein by a mammalian cell in culture, comprising: (a) providing a mammalian cell that produces a protein of interest, and (b) culturing said mammalian cell in a medium comprising a valproic acid compound selected from the group consisting of: (i) valproic acid, (ii) salts of valproic acid, and (iii) a combination of valproic acid and salts of valproic acid.

2. The process according to claim 1 wherein production of said protein in the presence of a valproic acid compound is enhanced compared to production of said protein in the absence of a valproic acid compound.

3. The process according to claim 1 wherein production of said protein in the presence of a valproic acid compound is enhanced compared to production of said protein in the presence of butyric acid or sodium butyrate.

4. The process according to claims 1 to 3 wherein said valproic acid compound is present at a concentration of between 0.002 mmol/l and 200 mmol/l, preferably between 0.01 mmol/l and 50 mmol/l and most preferably between 0.05 mM and 12 mmol/l.

5. The process of claims 1 to 3 where the mammalian cell is a HEK 293 cell and wherein said valproic acid compound is present at a concentration of 3.8 mmol/l.

6. The process of claims 1 to 3 where the mammalian cell is a CHO cell and wherein said valproic acid compound is present at a concentration of 0.5 mmol/l.

7. The process of claims 1 to 3, where the mammalian cell is a HEK 293 cell and wherein said valproic acid compound is present at a concentration of between 0.1 mmol/l and 12 mmol/l.

8. The process of claims 1 to 3, where the cell is a CHO cell and wherein said valproic acid compound is present at a concentration of between 0.05 mmol/l and 5 mmol/l.

9. The process of claims 1 to 8 where the valproic acid compound is added at a cell density of at least 1 million cells per ml.

10. The process of claims 1 to 8 where the valproic acid compound is added at a cell density of at least 2 million cells per ml.

11. The process of claims 1 to 8 where the valproic acid compound is added at a cell density of at least 4 million cells per ml.

12. The process of claims 1 to 11 where a valproic acid compound is added in combination with an inhibitor of DNA methyltransferases.

13. The process of claims 1 to 11 where a valproic acid compound is added in combination with a compound selected from the group consisting of azacytidine, RG108, decitabine.

14. The process of claims 1, 2, 3, 5, 6, 7, 8 where said mammalian cell is cultivated at a temperature of 37 degrees Celsius.

15. The process of claims 1, 2, 3, 5, 6, 7, 8 where said mammalian cell is cultivated at temperature of 31 degrees Celsius.

16. The process of claims 1, 2, 3, 5, 6, 7, 8 where said mammalian cell is cultivated at a temperature of between 28 degrees Celsius and 33 degrees Celsius.

17. The process according to claims 1 to 13 wherein said valproic acid compound is added at the beginning of the production phase.

18. The process according to claims 1 to 13 wherein said valproic acid compound is added at the beginning of the production phase and again at intervals thereafter.

19. The process according to claims 1, 2, 3, 5, 6, 7, 8, 14, 15, 16, comprising first culturing said eukaryotic cell in a cell culture medium that does not contain a valproic acid compound, prior to culturing said eukaryotic cell in a medium containing a valproic acid compound.

20. The process according to claims 1, 2, 3, 5, 6, 7, 8, 14, 15, 16, where said eukaryotic cell is cultured in a medium comprising a valproic acid compound for at least ten days.

21. A two stage process for the production of a protein according to claims 1, 2, 3 comprising: (a) a first stage of growing mammalian cells which produce said protein in growth medium until a predetermined cell density has been obtained; and (b) adding a valproic acid compound to said cells.

22. A three stage process for the production of a protein according to claims 1, 2, 3 comprising: (a) a first stage of expanding mammalian cells in growth medium; and (b) transfecting said mammalian cells with a gene expression vector encoding the protein of interest; and (c) cultivating said mammalian cells after transfection in a medium which contains a valproic acid compound.

23. The process of claims 1, 2, 3, 21, 22 where said protein is produced by means of transient gene expression.

24. The process of claims 1, 2, 3, 21 where said protein is produced by means of a stable producer cell.

25. The process of claim 23 where the valproic acid compound is added within 24 hours after transfection.

26. The process of claim 23 where the valproic acid compound is added within 48 hours after transfection.

27. The process according to claims 1, 2, 3, 5, 6, 7, 8, 14, 15, 16, 19, 20, 21, 22 wherein said eukaryotic cell is selected from mouse myeloma B (NS0) cells, Per.C6 cells, Chinese Hamster Ovary (CHO) cells, Baby Hamster Kidney (BHK) cells and Human Embryonic Kidney (HEK 293) cells.

28. The process of claims 23, 25, 26 where the transfected cell is a HEK 293 cell or CHO cell.

29. The process of claim 24 where the stable producer cell is a HEK 293 cell, CHO cell or Baby Hamster Kidney cell.

30. The process according to claims 1, 2, 3, 21, 22, 23, 24 wherein said protein is selected from the group consisting of growth factors, hormones, enzymes, enzyme inhibitors, cytokines, lymphokines, interleukins, blood clotting factors and immunoglobulins.

31. A process according to claims 1, 2, 3, 21, 22, 23, 24 wherein said protein is an immunoglobulin.

32. A process according to claim 1, 2, 3, 21, 22, 23, 24 wherein said protein is an Fc-fusion protein.

Description:

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC

Reference to Sequence Listing

[0001]A paper copy of the Sequence Listing and a computer readable form (CRF) of the sequence listing, containing the file named "VPA.ST25.txt" which is 86 kilobytes in size, and which was created on Jul. 1, 2007 and last modified on Jul. 1, 2007, are herein incorporated by reference. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. section 1.822.

BACKGROUND OF THE INVENTION

[0002]It must be noted that as used herein and in the appended claims, the singular forms "a" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" or "the cell" includes a plurality ("cells" or "the cells"), and so forth. Moreover, the word "or" can either be exclusive in nature (i.e., either A or B, but not A and B together), or inclusive in nature (A or B, including A alone, B alone, but also A and B together). One of skill in the art will realize which interpretation is the most appropriate unless it is detailed by reference in the text as "either A or B" (exclusive "or") or "and/or" (inclusive "or").

[0003]A portion of the disclosure of this patent document contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the Patent and Trademark Office file or records, but otherwise reserves all copyright rights whatsoever. The patent owners can be contacted at hildinger@gmx.net.

[0004](1) Field of the Invention

[0005]This invention relates to a process for the production of a protein of interest by cell culture, where cells which produce the protein of interest are cultured in the presence of a valproic acid compound, where the valproic acid compound increases batch yield and batch titer--all else equal.

[0006]Culturing cells for the commercial production of proteins for diagnosis and therapy is a costly and time consuming process. The equipment required is expensive, and production cost are high. In order to provide commercially viable processes it is desirable to use cell lines which produce large quantities of product with each production run. However, most cells do not produce large quantities of desired product per se either because they do not produce a large quantity of product per unit of time (specific productivity) or because they do not survive long enough in the culture medium (time). Here, we identified that addition of a valproic acid compound to the culture medium increases batch yield and titer--all else equal.

[0007](2) Description of Related Art

[0008]A number of chemical compounds are known which are able to act as enhancing agents of protein production in cell lines. Examples of those compounds are DMSO, urea derivatives and alkanoic acids or salts thereof. From amongst such enhancing agents, sodium butyrate has been the subject of intensive study in recent years. This compound has been added to cultures of a variety of naturally occurring and selected cell lines and has been shown to produce many morphological and biochemical modifications. At the molecular level, butyrate is believed to cause hyperacetylation of histones by inhibiting histone deacetylase (histone deacetylase inhibitor; HDAC inhibitor). Generally, butyrate appears to modify gene expression, and in almost all cases its addition to cells in culture appears to arrest cell growth.

[0009]Several patents have been filed around the use of alkanoic acids in general and sodium butyrate in particular in the context of enhanced production of a protein of interest, e.g., U.S. Pat. Nos. 5,681,718, 6,117,652, 6,740,505 to name a few. However, the use of a valproic acid compound in the context of enhancing production of a protein has not yet been published in prior art.

[0010]Valproic acid is indicated for the treatment of epilepsy (anticonvulsant) and is commercially available under the brand names Depakene, Valproate, Valrelease. It is used as a sole or adjunctive therapy in the treatment of simple or complex absence seizures, including petit mal, and is useful in primary generalized seizures with tonic-clonic manifestations. Valproic acid may also be used adjunctively in patients with multiple seizure types which include either absence or tonic-clonic seizures. Valproic acid also has efficacy as a mood stabilizer in bipolar disorder.

BRIEF SUMMARY OF THE INVENTION

[0011](1) Substance or General Idea of the Claimed Invention

[0012]Context: In a first aspect the present invention provides a process for the production of a protein of interest which comprises culturing cells which produce said protein in the presence of a valproic acid compound, which enhances protein production, wherein the valproic acid compound is present at a concentration at which production of said protein is enhanced.

[0013]In a further aspect, the invention provides a process for obtaining a protein by cell culture which comprises the steps of (1) culturing cells which produce said protein in the presence of a valproic acid compound, which enhances protein production, wherein the valproic acid compound is present at a concentration at which production of said protein is enhanced, (2) continuing said culture until said protein accumulates, and optionally (3) isolating said protein.

[0014]In yet another aspect, the present invention teaches that production of a protein in the presence of a valproic acid compound is enhanced compared to production of the same protein in the absence of a valproic acid compound.

[0015]In yet another aspect, the present invention teaches that production of a protein in the presence of a valproic acid compound is enhanced compared to production of the same protein in presence of butyric acid or sodium butyrate--assuming that the process conditions in both instances are optimized for the corresponding substances.

[0016]Nature of the valproic acid compound. The valproic acid compound can be valproic acid, a salt of valproic acid, or a combination of valproic acid and salt(s) of valproic acid.

[0017]Preferably, the valproic acid compound is valproic acid or a salt thereof, in particular an alkali metal salt, e.g. sodium salt.

[0018]Concentration of the valproic acid compound. The valproic acid compound may be present in the culture medium at a concentration of between 0.002 mmol/l and 200 mmol/l, preferably between 0.01 mmol/l and 50 mmol/l and most preferably between 0.05 mM and 12 mmol/l. In case the cell is a HEK 293 cell, the preferred final concentration of the valproic acid compound is between 0.1 mmol/and 12 mmol/l and the most preferred final concentration of the valproic acid compound is 3.8 mmol/l. In case the cell is a CHO cell, the preferred final concentration of the valproic acid compound is between 0.05 mmol/and 5 mmol/l and the most preferred final concentration of the valproic acid compound is 0.5 mmol/l.

[0019]It will be appreciated, however, that the concentration of the valproic acid compound employed may be varied depending on the particular cell line being cultured. The most appropriate concentration of a valproic acid compound for any particular cell line may need to be determined by appropriate small scale tests beforehand in accordance with conventional practice--without undue effort.

[0020]Cell density at time of valproic acid compound addition. In another aspect of the present invention, the valproic acid compound can be added at different cell densities such as at least 1 million cells per ml, preferably at least 2 million cells per ml and most preferably at least 4 million cells per ml. The exact cell density at which the valproic acid compound should be added depends on several factors:

(1) The valproic acid compound concentration: The higher the cell density, the higher the final concentration of valproic acid.(2) The medium: Not all media are able to sustain cell viability above certain cell densities.(3) The cell line: Different cell lines have different requirements in terms of cell density for cultivation.

[0021]All else equal, a higher cell density should be preferred as higher cell densities at a given specific productivity will result in higher batch titers. The most appropriate cell density at the time of valproic acid addition for any particular cell line or process may need to be determined by appropriate small scale tests beforehand in accordance with conventional practice--without undue effort.

[0022]Additional compounds: In some embodiments, additional compounds are added in combination with the valproic acid compound where said additional compounds act synergistically with the valproic acid compound. Such additional compounds are--without limitation--IDMTs (inhibitors of DNA methyltransferases) such as azacytidine, RG108, decitabine.

[0023]Temperature: In yet another aspect of the present invention, the mammalian cells are cultivated at a temperature of 37 degrees Celsius in the presence of a valproic acid compound, where the concentration of the valproic acid compound is sufficient to enhance protein production. In some embodiments, cells are cultivated at temperatures below 37 degrees Celsius, in a range of 28 degrees to 33 degrees Celsius and preferably at 31 degrees Celsius or 32 degrees Celsius. It has been shown in the literature that lowering the cultivation temperature can result in higher batch yields [7]. This is also applicable to processes where a valproic acid compound is present in the cultivation medium.

[0024]Frequency of valproic acid compound addition to the cells: In some embodiments, the valproic acid compound is added only once to the cells; in other embodiments, the valproic acid compound is added more than once to the cells, for example every other day.

[0025]Timing of valproic acid compound addition to the cells: In the process according to the invention, the valproic acid compound may be added to the culture medium at, before or after addition of the cells to the culture medium. If desired more than one addition of the valproic acid compound may be employed. Thus, for example, it may be desirable to add the valproic acid compound at the beginning of the culture and then to add more valproic acid compound as the culture proceeds, providing of course that the addition is closely controlled such that the concentration of the valproic acid compound does not go beyond that which is likely to reduce the cell growth rate or cell viability. Typically in this aspect of the invention the cells are grown in culture to provide the levels of biomass required for efficient protein production. The valproic acid compound may be included in the growth medium if desired but in some circumstances, however, it may be desirable to grow the cells in the absence of a valproic acid compound, for instance, when production of the protein has a deleterious effect on the cells. Thus cells may be grown to densities at or approaching maximum cell density in the case of suspension cultures, or to or approaching confluence in the case of adherent cell lines, at which stage they may be transferred to a production medium and the valproic acid compound added at a concentration which enhances protein production but which does not significantly reduce cell viability.

[0026]The valproic acid compound may also be used to increase protein production in cells which have stopped growing exponentially and are in stationary or decline phases of growth. This is particularly advantageous when the desired protein is only produced by the cell when it is in these last two phases. Thus in another aspect the invention provides a process for the production of a protein which comprises maintaining cells which produce said protein in culture in the presence of a valproic acid compound which enhances protein production wherein the valproic acid compound is present at a concentration at which production of said protein is enhanced but which does not significantly reduce cell viability (e.g. is substantially non-toxic to the cells).

[0027]In yet another aspect, the invention provides a process for the production of a protein which comprises a first stage in which cells which produce said protein, are grown in growth medium until a predetermined cell density has been obtained followed by a second stage in which said cells are maintained in the presence of a valproic acid compound which enhances protein production wherein said valproic acid compound is present at a concentration at which production of said protein is enhanced but at which cell growth is inhibited without significantly reducing cell viability. In said second stage, the cells may still be maintained in the growth medium, where valproic acid has been added to the growth medium (transforming the growth medium into a production medium), or the growth medium might be exchanged with a production medium comprising a valproic acid compound, or the growth medium might be modified, e.g., by adding additional compounds--including a valproic acid compound--to become a production medium.

[0028]The growth medium may contain a valproic acid compound which enhances production of the protein though preferably does not. The predetermined cell density to which the cells are grown in the first stage is preferably a cell density suitable for efficient protein production such as a density approaching maximum cell density for the culture or approaching confluence. The concentration of a valproic acid compound used in the second state will generally be as described previously but in particular will be such to arrest cell growth, prolong the period of cell viability and yield an increased level of overall protein production.

[0029]In the context of transient gene expression, where the gene expression vector encoding the protein of interest is introduced into the cell by means of transfection, the valproic acid compound is added within 48 hours of transfection and more preferably within 24 hours after transfection. In that instance, the cells are expanded in growth medium prior to transfection, transferred into transfection medium for the transfection, and then transferred into production medium for protein production. In such a setting, only the production medium might comprise the valproic acid compound. Yet, in some embodiments, also the transfection medium might comprise a valproic acid compound.

[0030]Stable cell line vs. transient gene expression: In another aspect, the cell which produces the protein of interest has the corresponding transgene stably integrated in its genome, i.e., the cell is a stable producer cell. In yet another aspect, the expression cassette encoding the genetic information for the protein of interest is introduced into the cell by means of transfection. For example, the cells to be transfected will be expanded in a first stage in growth medium, transfected with a gene expression vector encoding the protein of interest in a second stage in transfection medium and cultivated in production medium after transfection for 1 day to 3 weeks or longer. The valproic acid compound might be added to the growth, transfection and production medium, preferably only to the transfection and production medium and most preferably to the production medium only. Transfection methods have been described in detail in prior art (see also the definition section). Most preferably, transfection is performed with 25-kd linear polyethyleneimine.

[0031]Cell type: The cell which is used for the production of the protein of interest, be it by means of stable integration or by transient gene expression, can be any mammalian cell, preferably myeloma B (NS0) cells, Per.C6 cells, Chinese Hamster Ovary (CHO) cells, Baby Hamster Kidney (BHK) cells or Human Embryonic Kidney (HEK 293) cells, most preferably CHO or HEK 293 cells.

[0032]This embodiment of the invention is particularly applicable to cell lines which are intermediate or low producers of a desired protein, making it possible to substantially increase protein production by use of a valproic acid compound at an appropriate concentration.

[0033]Nature of the protein produced: The protein produced by the process is preferably an immunoglobulin or an FC-fusion protein. However, any other protein of interest can be produced by the teachings of the present invention--including, but not limited to, growth factors, hormones, enzymes, enzyme inhibitors, cytokines, lymphokines, interleukins, blood clotting factors, fusion proteins.

[0034]Any suitable culture procedure and culture medium may be used to culture the cells in the process of the invention. Suitable culture procedures are well known and understood by workers in the cell culture art. Both serum supplemented and serum free media may be used. Batch and continuous fermentation procedures, suspension and adherent, e.g. microcarrier culture methods and stirred tank and airlift fermenters may be used as appropriate having regard to cell type.

[0035]The production of the protein during the culture may be monitored by general assay techniques such as enzyme linked immunosorbent assay or immunoradiometric assay adapted for the particular protein in question.

[0036]The desired protein may, if required, be isolated from the cell culture by conventional separation techniques. Thus, for example, protein in the culture medium may be separated from cells by centrifugation, the supernatant containing the protein being collected after concentration (for example by ultrafiltration). The protein so obtained may be further purified, if desired, using conventional protein purification methods. Where the desired protein is not secreted into the culture medium during the culture it may obtained by rupture of the cells, then subsequent processing as just described.

[0037](2) Advantages of the Invention Over Prior Approaches

[0038]Usefulness of the Present Invention

[0039]The advantage of adding a valproic acid compound to a culture system for the production of a protein of interest is that protein production can be substantially increased for a very small outlay in expenditure, and little if any alteration to culturing techniques. Moreover, the inventors show that adding a valproic acid compound enhances protein production more compared to adding sodium butyrate. Last, but not least, valproic acid is approved by the FDA for human usage, which makes it a safe compound to use for cell culture purposes.

[0040]Novelty of the Present Invention

[0041]As mentioned above, several patents have been filed around the use of alkanoic acids in general and sodium butyrate in particular in the context of enhanced production of a protein of interest. However, the use of a valproic acid compound in the context of enhancing production of a protein has not yet been published in prior art. And it cannot be assumed (without undue effort) that any alkanoic acid (including valproic acid) behaves similarly as sodium butyrate--unless one performs the necessary experiments. In particular, it cannot be assumed that adding a valproic acid increases batch yield more than adding sodium butyrate. The inventors--for the first time--show that adding a valproic acid compound enhances protein production more compared to adding sodium butyrate. This is also novel and has not been shown in prior art.

[0042]Non-Obviousness of the Present Invention

[0043]Whereas sodium butyrate has been extensively studied in the context of enhancing protein production, it is not obvious that all other alkanoic acids will have a similar effect. Moreover, the inventors show that valproic acid compounds enhance protein production more than sodium butyrate, which is also not obvious--unless one conducts the necessary experiments. Moreover, based on the use of valproic acid to treat seizures in humans, it seems quite a stretch to assume that it also has benefits in the production of recombinant proteins by mammalian cells in vitro.

[0044]Furthermore, given the high commercial interest in increasing batch yields and lowering production cost, and the ongoing research on the use of alkanoic acids to enhance protein production, the use of a valproic acid compound in that context would have been described already if it were obvious, particularly as valproic acid is an economically feasible alternative to sodium butyrate.

DETAILED DESCRIPTION OF THE INVENTION

[0045]The practice of the present invention will employ, unless otherwise indicated, conventional methods of virology, microbiology, molecular biology and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature; see, e.g., Sambrook, et al. Molecular Cloning: A Laboratory Manual (Current Edition); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., Current Edition); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., Current Edition); Transcription and Translation (B. Hames & S. Higgins, eds., Current Edition); CRC Handbook of Parvoviruses, vol. I & II (P. Tijessen, ed.); Fundamental Virology, 2nd Edition, vol. I & II (B. N. Fields and D. M. Knipe, eds.)

[0046](1) Definitions

[0047]In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.

[0048]For purposes of this invention, the term "enhancing agent" means a compound whose presence in a culture medium increases batch yield compared to the same process and culture medium in the absence of said compound.

[0049]For purposes of this invention, the term "valproic acid compound" or "VPA" means valproic acid, a valproic acid salt, a combination of valproic acid and valproic acid salt(s) or any other valproic acid derivative. Unless noted otherwise, VPA was purchased from Sigma-Aldrich Chemie GmbH, Industriestrasse 25, CH-9471 Buchs SG, Catalog Number P4543, and used in the experiments and embodiments of the present invention as described in the corresponding sections.

[0050]For purposes of this invention, the term "protein" means a polypeptide (native [i.e., naturally-occurring] or mutant), oligopeptide, peptide, or other amino acid sequence. As used herein, "protein" is not limited to native or full-length proteins, but is meant to encompass protein fragments having a desired activity or other desirable biological characteristics, as well as mutants or derivatives of such proteins or protein fragments that retain a desired activity or other biological characteristic including peptoids with nitrogen based backbone. Mutant proteins encompass proteins having an amino acid sequence that is altered relative to the native protein from which it is derived, where the alterations can include amino acid substitutions (conservative or non-conservative), deletions, or additions (e.g., as in a fusion protein). "Protein" and "polypeptide" are used interchangeably herein without intending to limit the scope of either term.

[0051]For purposes of this invention, "amino acid" refers to a monomeric unit of a peptide, polypeptide, or protein. There are twenty amino acids found in naturally occurring peptides, polypeptides and proteins, all of which are L-isomers. The term also includes analogs of the amino acids and D-isomers of the protein amino acids and their analogs.

[0052]For purposes of this invention, by "DNA" is meant a polymeric form of desoxyribonucleotides (adenine, guanine, thymine, or cytosine) in double-stranded or single-stranded form, either relaxed or supercoiled, either linear or circular. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes single- and double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure of particular DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the non-transcribed strand of DNA (i.e., the strand having the sequence homologous to the mRNA). The term captures molecules that include the four bases adenine (A or a), guanine (G or g), thymine (T or t), or cytosine (C or c), as well as molecules that include base analogues which are known in the art.

[0053]For purposes of this invention, "polynucleotide" as used herein means a polymeric form of nucleotides of any length, either ribonucleotides or desoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, the term includes double- and single-stranded DNA, as well as, double- and single-stranded RNA. It also includes modifications, such as methylation or capping, and unmodified forms of the polynucleotide.

[0054]For the purpose of describing the relative position of nucleotide sequences in a particular nucleic acid molecule throughout the instant application, such as when a particular nucleotide sequence is described as being situated "upstream," "downstream," "5'," or "3'" relative to another sequence, it is to be understood that it is the position of the sequences in the non-transcribed strand of a DNA molecule that is being referred to as is conventional in the art.

[0055]For purposes of this invention, a "gene sequence" or "coding sequence" or "protein coding sequence" or "open reading frame" or "cDNA" or a sequence which "encodes" a particular protein, is a nucleic acid composition which is transcribed into RNA (in the case of DNA) and potentially translated (in the case of mRNA) into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory control elements. The boundaries of the gene are determined by a start codon at the 5' (amino) terminus and potentially a translation stop codon at the 3' (carboxy) terminus. A gene sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence, which is a particular species of regulatory control element, will usually be located 3' to the protein coding sequence.

[0056]For purposes of this invention, by the term "transgene" is meant a nucleic acid composition made out of DNA, which encodes a peptide, oligopeptide or protein. The transgene may be operatively linked to regulatory control elements in a manner which permits transgene transcription, translation and/or ultimately directs expression of a product encoded by the expression cassette in the producer cell, e.g., the transgene is placed into operative association with a promoter and enhancer elements, as well as other regulatory control elements, such as introns or polyA sequences, useful for its regulation. The composite association of the transgene with its regulatory sequences (regulatory control elements) is referred to herein as a "minicassette", "expression cassette", "transgene expression cassette", or "minigene". The exact composition of the expression cassette will depend upon the use to which the resulting (mini)gene transfer vector will be put and is known to the artisan (Sambrook 1989, Lodish et al. 2000). When taken up by a target cell, the expression cassette as part of the recombinant vector genome may remain present in the cell as a functioning extrachromosomal molecule, or it may integrate into the cell's chromosomal DNA, depending on the kind of transfer vector used. Generally, a minigene may have a size in the range of several hundred base pairs up to about 30 kb.

[0057]For purposes of this invention, "heterologous" as it relates to nucleic acid compositions denotes sequences that are not normally joined together. Thus, a "heterologous" region of a nucleic acid composition is a segment of nucleic acid within or attached to another nucleic acid composition that is not found in association with the other molecule in nature. For example, a heterologous region of a nucleic acid composition could include a coding sequence flanked by sequences not found in association with the coding sequence in nature. Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., synthetic sequences having codons different from the native gene). Allelic variation or naturally occurring mutational events do not give rise to heterologous DNA, as used herein.

[0058]For purposes of this invention, "homology" or "homologous" refers to the percent homology between two polynucleotide moieties or two polypeptide moieties. The correspondence between the sequence from one moiety to another can be determined by techniques known in the art. Two DNA or two polypeptide sequences are "substantially homologous" to each other when at least about 80%, preferably at least about 90%, and most preferably at least about 95% of the nucleotides or amino acids match over a defined length of the molecules, as determined using methods in the art.

[0059]The techniques for determining amino acid sequence homology are well-known in the art. In general, "homology" (for amino acid sequences) means the exact amino acid to amino acid comparison of two or more polypeptides at the appropriate place, where amino acids are identical or possess similar chemical and/or physical properties such as charge or hydrophobicity. A so-termed "percent homology" then can be determined between the compared polypeptide sequences. The programs available in the Wisconsin Sequence Analysis Package (available from Genetics Computer Group, Madison, Wis.), for example, the GAP program, are capable of calculating homologies between two polypeptide sequences. In addition, the ClustalW algorithm is capable of performing a similar analysis. Other programs and algorithms for determining homology between polypeptide sequences are known in the art.

[0060]Homology for polynucleotides is determined essentially as follows: Two polynucleotides are considered to be "substantially homologous" to each other when at least about 80%, preferably at least about 90%, and most preferably at least about 95% of the nucleotides match over a defined length of the molecules, when aligned using the default parameters of the search algorithm BLAST 2.0. The BLAST 2.0 program is publicly available. The ClustalW algorithm can be utilized as well.

[0061]Alternatively, homology for polynucleotides can be determined by hybridization experiments. As used herein, a nucleic acid sequence or fragment (such as for example, primers or probes), is considered to selectively hybridize to a sequence 1, thus indicating "substantial homology", if such a sequence is capable of specifically hybridizing to the sequence 1 or a variant thereof or specifically priming a polymerase chain reaction: (i) under typical hybridization and wash conditions, such as those described, for example, in Maniatis, (Molecular Cloning: A Laboratory Manual, 2nd Edition, 1989) where preferred hybridization conditions are those of lesser stringency and more preferred, higher stringency; or (ii) using reduced stringency wash conditions that allow at most about 25-30% base pair mismatches, for example, 2×SSC, 0.1% SDS, at room temperature twice, for 30 minutes each; then 2×SSC, 0.1% SDS, 37° C., once for 30 minutes; the 2×SSC at room temperature twice, 10 minutes each or (iii) under standard PCR conditions or under "touch-down" PCR conditions.

[0062]For purposes of this invention, the term "cell" means any prokaryotic or eukaryotic cell, either ex vivo, in vitro or in vivo, either separate (in suspension) or as part of a higher structure such as but not limited to organs or tissues.

[0063]For purposes of this invention, the term "host cell" means a cell that can be transduced and/or transfected by an appropriate gene transfer vector. The nature of the host cell may vary from gene transfer vector to gene transfer vector.

[0064]For purposes of this invention, the term "producer cell" means a cell that is capable of producing a recombinant protein or protein of interest. The producer cell itself may be selected from any mammalian cell. Particularly desirable producer cells are selected from among any mammalian species, including, without limitation, cells such as HEK 293, A549, WEHI, 3T3, 10 T1/2, BHK, MDCK, COS 1, COS 7, BSC 1, BSC 40, BMT 10, VERO, WI38, HeLa, Saos, C2C12, L cells, HT1080, HepG2, CHO, NS0, Per.C6. The selection of the mammalian species providing the cells is not a limitation of this invention; nor is the type of mammalian cell, i.e., fibroblast, hepatocyte, tumor cell, etc. Frequently used producer cells or HEK 293 cells, BHK cells, NS0 cells, Per.C6 cells and CHO cells. Preferentially, a producer cell should be free of potential adventitious viruses.

[0065]For purposes of this invention, "transfection" is used to refer to the uptake of nucleic acid compositions by a cell. A cell has been "transfected" when an exogenous nucleic acid composition has crossed the cell membrane. A number of transfection techniques are generally known in the art. Such techniques can be used to introduce one or more nucleic acid compositions, such as a plasmid vector and other nucleic acid molecules, into suitable host cells. Frequently, cells are transfected with 25-kd linear polyethyleneimine. Other alternatives are transfection by means of electroporation, liposomes, dendrimers, or calcium phosphate.

[0066]For purposes of this invention, by "vector", "transfer vector", "gene transfer vector" or "nucleic acid composition transfer vector" is meant any element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virus capsid, virion, etc., which is capable of transferring and/or transporting a nucleic acid composition to a host cell, into a host cell and/or to a specific location and/or compartment within a host cell. Thus, the term includes cloning and expression vehicles, as well as viral and non-viral vectors and potentially naked or complexed DNA. However, the term does not include cells that produce gene transfer vectors such as retroviral packaging cell lines.

[0067]For purposes of this invention, the term "control elements", "regulatory sequences" or "regulatory control elements" refers collectively to promoter regions, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control elements need always be present as long as the selected coding sequence is capable of being replicated, transcribed and/or translated in an appropriate host cell. Sometimes, the entirety of control elements and coding sequence is referred to as "gene"; in other instances, "gene" only refers to the coding sequence. For purposes of this invention, "gene" refers to the entirety of control elements and coding sequence. Expression control elements include appropriate transcription initiation, termination, promoter and enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation signals, sequences that stabilize cytoplasmic mRNA, sequences that enhance translation efficacy (i.e., Kozak consensus sequence), sequences that enhance protein stability, and when desired, sequences that enhance protein processing and/or secretion. A great number of expression control elements, e.g., native, constitutive, inducible and/or tissue specific, are known in the art and may be utilized to drive expression of the gene, depending upon the type of expression desired. For eukaryotic cells, expression control elements typically include a promoter, an enhancer, such as one derived from an immunoglobulin gene, SV40, cytomegalovirus, etc., a polyadenylation sequence, and may include splice donor and acceptor sites. The polyadenylation sequence generally is inserted following the transgene sequences and before the 3' ITR sequence in rAAV vectors.

[0068]The regulatory sequences useful in the constructs of the present invention may also contain an intron, desirably located between the promoter/enhancer sequence and the gene. One possible intron sequence is derived from SV40, and is referred to as the SV40 T intron sequence. Another suitable regulatory sequence includes the woodchuck hepatitis virus post-transcriptional element. Still other methods may involve the use of a second internal promoter, an alternative splice signal, a co- or post-translational proteolytic cleavage strategy, among others which are known to those of skill in the art. Selection of these and other common vector and regulatory sequences are conventional, and many such sequences are available. See, e.g., Sambrook et al, and references cited therein at, for example, pages 3.18-3.26 and 16.17-16.27 and Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1989.

[0069]One of skill in the art may make a selection among these regulatory sequences without departing from the scope of this invention. Suitable promoter/enhancer sequences may be selected by one of skill in the art using the guidance provided by this application. Such selection is a routine matter and is not a limitation of the present invention.

[0070]For purposes of this invention, the term "promoter" means a regulatory sequence capable of binding RNA polymerase and/or a regulatory sequence sufficient to direct transcription. "Promoter" is also meant to encompass those promoter (or enhancer) elements for cell-type specific, tissue-specific and/or inducible (by external signals or agents) transcription; such elements may be located in the 5' or 3' regions of a native gene.

[0071]For purposes of this invention, the term "operative association" or "operative linkage" refers to an arrangement of elements or nucleic acid sequences wherein the compounds so described are configured so as to perform their intended function. Thus, (a) regulatory sequence(s) operably linked to a coding sequence is/are capable of effecting the expression of said coding sequence and is/are connected in such a way as to permit gene expression of the coding sequence when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence(s). The regulatory sequences need not be contiguous with the coding sequence, as long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence. "Operably linked" sequences include both expression control sequences that are contiguous with the coding sequences for the product of interest and expression control sequences that act in trans or at a distance to control the expression of the product of interest.

[0072]For purpose of this invention, the term "specific productivity" refers to the amount of the protein of interest that is produced by a single cell per day. For example a specific productivity of 20 pg/cell/day refers to the production of 20 pg of the protein of interest by a single cell within 24 hours.

[0073]For purpose of this invention, the term "batch" refers to the (specific lot of) protein molecules of interest produced in a single production run, i.e., under the same production conditions. Batch means a specific quantity of a drug or other material that is intended to have uniform character and quality, within specified limits, and is produced according to a single manufacturing order during the same cycle of manufacture.

[0074]For purpose of this invention, the term "lot" means a batch, or a specific identified portion of a batch, having uniform character and quality within specified limits; or, in the case of a drug product produced by continuous process, it is a specific identified amount produced in a unit of time or quantity in a manner that assures its having uniform character and quality within specified limits

[0075]For purpose of this invention, the term "batch yield" refers to the maximum amount (in grams) of the recombinant protein of interest produced by all of the mammalian cells in the culture batch together. For secreted proteins, the "batch yield" refers to the maximum amount of the recombinant protein of interest in the culture medium where the recombinant protein of interest is secreted into the medium by the mammalian cells present in the medium. For example, if a mammalian cell culture of 1 liter comprises 0.5 g of recombinant protein of interest in total, the batch yield is 500 mg and the batch titer is 500 mg/l. Thus, whereas the specific productivity refers to the production of recombinant protein by a single mammalian cell within one day, the batch yield refers to the maximum amount of recombinant protein produced by all the mammalian cells in the culture during the total time of the culture. "Volumetric yield" can be used as a synonym for "batch yield".

[0076]For purpose of this invention, the term "batch titer" refers to the maximum concentration (in grams per liter or milligrams per liter) of the recombinant protein of interest produced by all of the mammalian cells in the culture batch together. For secreted proteins, the "batch titer" refers to the maximum concentration of the recombinant protein of interest in the culture medium where the recombinant protein of interest is secreted into the medium by the mammalian cells present in the medium. For example, if a mammalian cell culture of 1 liter comprises 0.5 g of recombinant protein of interest in total, the batch yield is 0.5 grams and the batch titer is 0.5 g/l. Thus, whereas the specific productivity refers to the production of recombinant protein by a single mammalian cell within one day, the batch titer refers to the maximum concentration of recombinant protein produced by all the mammalian cells in the culture during the total time of the culture. The batch titer could also be defined as batch yield divided by culture volume.

[0077]For purpose of this invention, "growth medium" refers to a cell culture medium that promotes cell growth and division--leading to an increase in biomass as it relates to the cells. Optimally, a growth medium allows for a fast increase in biomass and supports cell growth to high cell densities.

[0078]For purpose of this invention, "transfection medium" refers to a cell culture medium that is suitable for transfection. Transfection media do not necessarily support cell growth or production. For example, RPMI can be used as transfection medium, but is not well suited for cell growth or production. An optimal transfection medium does not interfere with the transfection process, e.g., it does not contain inhibitors that inactivate the transfection reagent.

[0079]For purpose of this invention, "production medium" refers to a cell culture medium that promotes production of the protein of interest. A production medium does not necessarily support cell growth. Furthermore, one cannot necessarily transfect in production media, or only at a low transfection efficacy. An optimal production medium has the following characteristics: It sustains cell viability at a high cell density and results in high specific productivity for an extended period of time.

[0080](2) General Methods

[0081]The practice of the present invention will employ, unless otherwise indicated, conventional methods of microbiology, molecular biology and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature; see, e.g., Sambrook, et al. Molecular Cloning: A Laboratory Manual (Current Edition); DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., Current Edition); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., Current Edition); Transcription and Translation (B. Hames & S. Higgins, eds., Current Edition); CRC Handbook of Parvoviruses, vol. I & II (P. Tijessen, ed.); Fundamental Virology, 2nd Edition, vol. I & II (B. N. Fields and D. M. Knipe, eds.)

[0082]Unless otherwise noted, all experiments (examples) described in the following paragraphs were performed in triplicates.

EXAMPLE 1

Influence of VPA Addition on Batch Titers

[0083]10 million suspension-adapted HEK293E cells [1] were resuspended in 0.5 ml of Ex-Cell 293 HEK 293 serum-free medium with 4 mmol/l L-glutamine (Cat. No. 14571C-1000 M; Lot No. 6A0093; SAFC Biosciences, Lenexa, Kans., USA, "Ex-Cell" medium; note: "Ex-Cell" medium--as used herein--refers to a medium comprising 4 mmol/l L-glutamine) in a 50-ml filter tube each (TPP AG, Trasadingen, Switzerland; Cat. No. 87050, Lot 20050174). In a separate set, 10 million suspension-adapted CHO DG44 cells [6] were resuspended in 0.5 ml of ProCHO5 serum-free medium with 4 mmol/l L-glutamine and 0.68 g/l hypoxanthine and 0.194 g/l thymidine (BioWhittaker (Lonza), Belgium; Cat. No. BE12-766Q; Lot No. 7 MB0040; "ProCHO5" medium; note: "ProCHO5" medium--as used herein--refers to a medium comprising 4 mmol/l L-glutamine and 0.68 g/l hypoxanthine and 0.194 g/l thymidine) in a 50-ml filter tube each (TPP AG, Trasadingen, Switzerland; Cat. No. 87050, Lot 20050174).

[0084]Then 12.5 μg plasmid DNA at a concentration of 1 μg/μl was added in each tube with the following composition

[0085]50% (6.25 μg) p-LC (SEQ ID NO: 1)

[0086]50% (6.25 μg) p-HC (SEQ ID NO: 3).

[0087]Plasmid p-LC comprises the genetic information for the production of the light chain of an IgG antibody, plasmid p-HC comprises the genetic information for the production of the heavy chain of an IgG antibody. The particular vector constructs used in the present invention can also be obtained from the inventors. The inventors can be contacted via e-mail at hildinger@gmx.net. Moreover, commercial services do exist that produce any desired nucleotide sequence even comprising several kilobase pairs including complete expression cassettes in plasmid backgrounds (e.g., Invitrogen, Carlsbad, USA; Geneart, Germany.) Thus, providing the genetic sequence information of the plasmids should enable one of ordinary skill in the art to order the plasmids of the present invention at one of the commercial services listed.

[0088]After shortly mixing the DNA with the cells by gentle shaking, 25 μg of 25-kd linear polyethyleneimine ("PEI"; Polysciences, Eppelheim, Germany; [2]) were added at a concentration of 1 μg/μl to each of the tubes. After shortly mixing by gentle shaking, the filter tubes containing the cells together with the DNA and PEI were transferred into an orbital shaker (Kuhner Shaker Cabinet ISF-4-W, "Kuhner shaker", Kuhner AG, Birsfelden, Switzerland), and the cells were incubated for 4 hours at 37° C. in a 5% CO₂ atmosphere under shaking at 180 rpm. After that time, 4.5 ml of Ex-Cell medium were added to each of the filter tubes containing HEK 293 cells and 4.5 ml of Pro-CHO5 were added to each of the filter tubes containing CHO DG44 cells. Then, valproic acid was added to each of the tubes at the corresponding final concentration. The cells then were returned into the Kuhner shaker and incubated at 37° C. in a 5% CO₂ atmosphere under shaking at 180 rpm. Five days post transfectionem, 200 μl of supernatant was removed from the cells in order to determine the antibody titer via ELISA.

[0089]The ELISA was performed as published in prior art [3]. In short, Goat anti-human kappa light chain IgG (Biosource) was used for coating the ELISA-plates, and with AP-conjugated goat anti-human gamma chain IgG (Biosource) the synthesized IgG1 was detected. NPP was used as a substrate for the alkaline phosphatase. Absorption was measured at 405 nm against 490 nm using a microplate reader (SPECTRAmax®340; Molecular Devices, Palo Alto, Calif., USA).

[0090]The following results were obtained at day 5 after transfection for HEK293E cells with standard deviation given as +/-values:

TABLE-US-00001 Final VPA concentration Batch titer (mg/l) Fold increase over control 0 mmol/l (control) 15.0 ± 1.8 1 1.3 mmol/l 33.0 ± 4.3 2.2 2.5 mmol/l 61.8 ± 5.7 4.1 4 mmol/l 79.5 ± 3.6 5.3 5 mmol/l 64.5 ± 6.5 4.3 7.5 mmol/l 27.0 ± 3.1 1.8 11.3 mmol/l 13.5 ± 2.1 0.9

[0091]As one can see, batch titers are increasing when adding valproic acid. Only at very high valproic acid concentrations, batch titers are decreasing again due to lower cell viability. The optimal final concentration for HEK293E cells is at around 4 mmol/l.

[0092]The following results were obtained at day 5 after transfection for CHO cells with standard deviation given as +/-values:

TABLE-US-00002 Final VPA concentration Batch titer (mg/l) Fold increase over control 0 mmol/l (control) 7.4 ± 1.2 1 0.05 mmol/l 7.4 ± 1.1 1 0.1 mmol/l 10.0 ± 1.5 1.3 0.2 mmol/l 13.7 ± 1.2 1.8 0.5 mmol/l 16.3 ± 1.3 2.1 1 mmol/l 14.4 ± 1.2 1.9 2 mmol/l 10.0 ± 0.9 1.3 3 mmol/l 7.4 ± 1.2 1.0

[0093]As one can see, batch titers are increasing when adding valproic acid. Only at very high valproic acid concentrations, batch titers are decreasing again due to lower cell viability. The optimal final concentration for CHO DG44 cells is at around 0.5 mmol/l.

EXAMPLE 2

Influence of Temperature on Batch Titer and Comparison with Sodium Butyrate

[0094]To analyze the influence of temperature on batch titer, 10 million suspension-adapted HEK293E cells [1] were resuspended in 0.5 ml of Ex-Cell 293 HEK 293 serum-free medium with 4 mmol/l L-glutamine (Cat. No. 14571C-1000M; Lot No. 6A0093; SAFC Biosciences, Lenexa, Kans., USA, "Ex-Cell" medium; note: "Ex-Cell" medium--as used herein--refers to a medium comprising 4 mmol/l L-glutamine) in a 50-ml filter tube each (TPP AG, Trasadingen, Switzerland; Cat. No. 87050, Lot 20050174). In a separate set, 10 million suspension-adapted CHO DG44 cells [6] were resuspended in 0.5 ml of ProCHO5 serum-free medium with 4 mmol/l L-glutamine and 0.68 g/l hypoxanthine and 0.194 g/l thymidine (BioWhittaker (Lonza), Belgium; Cat. No. BE12-766Q; Lot No. 7 MB0040; "ProCHO5" medium; note: "ProCHO5" medium--as used herein--refers to a medium comprising 4 mmol/l L-glutamine and 0.68 g/l hypoxanthine and 0.194 g/l thymidine) in a 50-ml filter tube each (TPP AG, Trasadingen, Switzerland; Cat. No. 87050, Lot 20050174).

[0095]Then 12.5 μg plasmid DNA at a concentration of 1 μg/μl was added in each tube with the following composition

[0096]50% (6.25 μg) p-LC (SEQ ID NO: 1)

[0097]50% (6.25 μg) p-HC (SEQ ID NO: 3).

[0098]Plasmid p-LC comprises the genetic information for the production of the light chain of an IgG antibody, plasmid p-HC comprises the genetic information for the production of the heavy chain of an IgG antibody. The particular vector constructs used in the present invention can also be obtained from the inventors. The inventors can be contacted via e-mail at hildinger@gmx.net. Moreover, commercial services do exist that produce any desired nucleotide sequence even comprising several kilobase pairs including complete expression cassettes in plasmid backgrounds (e.g., Invitrogen, Carlsbad, USA; Geneart, Germany.) Thus, providing the genetic sequence information of the plasmids should enable one of ordinary skill in the art to order the plasmids of the present invention at one of the commercial services listed.

[0099]After shortly mixing the DNA with the cells by gentle shaking, 25 μg of 25-kd linear polyethyleneimine ("PEI"; Polysciences, Eppelheim, Germany; [2]) were added at a concentration of 1 μg/μl to each of the tubes. After shortly mixing by gentle shaking, the filter tubes containing the cells together with the DNA and PEI were transferred into an orbital shaker (Kuhner Shaker Cabinet ISF-4-W, "Kuhner shaker", Kuhner AG, Birsfelden, Switzerland), and the cells were incubated for 4 hours at 37° C. in a 5% CO₂ atmosphere under shaking at 180 rpm. After that time, 4.5 ml of Ex-Cell medium were added to each of the filter tubes containing HEK 293 cells and 4.5 ml of Pro-CHO5 were added to each of the filter tubes containing CHO DG44 cells. Then, valproic acid was added to the corresponding tubes at the corresponding final concentration, and sodium butyrate (Cat. No. B5887-1G; Sigma-Aldrich Chemie GmbH, Industriestrasse 25, CH-9471 Buchs SG) was added to the corresponding tubes at the corresponding final concentrations. The cells then were returned into the Kuhner shaker. Half the tubes were incubated at 37° C. in a 5% CO₂ atmosphere under shaking at 180 rpm, the other half was incubated at 31° C. Five days post transfectionem, 200 μl of supernatant was removed from the cells in order to determine the antibody titer via ELISA.

[0100]The ELISA was performed as published in prior art [3]. In short, Goat anti-human kappa light chain IgG (Biosource) was used for coating the ELISA-plates, and with AP-conjugated goat anti-human gamma chain IgG (Biosource) the synthesized IgG1 was detected. NPP was used as a substrate for the alkaline phosphatase. Absorption was measured at 405 nm against 490 nm using a microplate reader (SPECTRAmax®340; Molecular Devices, Palo Alto, Calif., USA).

[0101]The following results were obtained at day 5 after transfection for HEK293E cells with standard deviation given as +/-values:

TABLE-US-00003 Batch titer increase Batch titer increase Final concentration at 31° C. at 37° C. Control (no VPA; no sodium 1 1 butyrate) 3.8 mmol/l VPA 5.3 ± 0.4 5.3 ± 0.5 3 mmol/l sodium butyrate 4.8 ± 0.4 4.8 ± 0.4

[0102]As one can see, batch titers are increased both at 31° C. and 37° C., with VPA showing a slightly better performance than sodium butyrate at day 5 after transfection.

[0103]The following results were obtained at day 5 after transfection for CHO cells with standard deviation given as +/-values:

TABLE-US-00004 Batch titer increase Batch titer increase Final concentration at 31° C. at 37° C. Control (no VPA; no sodium 1 1 butyrate) 3.8 mmol/l VPA 1.5 ± 0.1 2.1 ± 0.1 3 mmol/l sodium butyrate 1.3 ± 0.1 1.9 ± 0.1

[0104]As one can see, batch titers are increased both at 31° C. and 37° C., with VPA showing a slightly better performance than sodium butyrate at day 5 after transfection.

EXAMPLE 3

Multiple VPA Additions

[0105]10 million suspension-adapted HEK293E cells [1] were resuspended in 0.5 ml of Ex-Cell 293 HEK 293 serum-free medium with 4 mmol/l L-glutamine (Cat. No. 14571C-1000M; Lot No. 6A0093; SAFC Biosciences, Lenexa, Kans., USA, "Ex-Cell" medium; note: "Ex-Cell" medium--as used herein--refers to a medium comprising 4 mmol/l L-glutamine) in a 50-ml filter tube each (TPP AG, Trasadingen, Switzerland; Cat. No. 87050, Lot 20050174). In a separate set, 10 million suspension-adapted CHO DG44 cells [6] were resuspended in 0.5 ml of ProCHO5 serum-free medium with 4 mmol/l L-glutamine and 0.68 g/l hypoxanthine and 0.194 g/l thymidine (BioWhittaker (Lonza), Belgium; Cat. No. BE12-766Q; Lot No. 7 MB0040; "ProCHO5" medium; note: "ProCHO5" medium--as used herein--refers to a medium comprising 4 mmol/l L-glutamine and 0.68 g/l hypoxanthine and 0.194 g/l thymidine) in a 50-ml filter tube each (TPP AG, Trasadingen, Switzerland; Cat. No. 87050, Lot 20050174).

[0106]Then 12.5 μg plasmid DNA at a concentration of 1 μg/μl was added in each tube with the following composition

[0107]50% (6.25 μg) p-LC (SEQ ID NO: 1)

[0108]50% (6.25 μg) p-HC (SEQ ID NO: 3).

[0109]Plasmid p-LC comprises the genetic information for the production of the light chain of an IgG antibody, plasmid p-HC comprises the genetic information for the production of the heavy chain of an IgG antibody. The particular vector constructs used in the present invention can also be obtained from the inventors. The inventors can be contacted via e-mail at hildinger@gmx.net. Moreover, commercial services do exist that produce any desired nucleotide sequence even comprising several kilobase pairs including complete expression cassettes in plasmid backgrounds (e.g., Invitrogen, Carlsbad, USA; Geneart, Germany.) Thus, providing the genetic sequence information of the plasmids should enable one of ordinary skill in the art to order the plasmids of the present invention at one of the commercial services listed.

[0110]After shortly mixing the DNA with the cells by gentle shaking, 25 μg of 25-kd linear polyethyleneimine ("PEI"; Polysciences, Eppelheim, Germany; [2]) were added at a concentration of 1 μg/μl to each of the tubes. After shortly mixing by gentle shaking, the filter tubes containing the cells together with the DNA and PEI were transferred into an orbital shaker (Kuhner Shaker Cabinet ISF-4-W, "Kuhner shaker", Kuhner AG, Birsfelden, Switzerland), and the cells were incubated for 4 hours at 37° C. in a 5% CO₂ atmosphere under shaking at 180 rpm. After that time, 4.5 ml of Ex-Cell medium were added to each of the filter tubes containing HEK 293 cells and 4.5 ml of Pro-CHO5 were added to each of the filter tubes containing CHO DG44 cells. Then, valproic acid was added to the corresponding tubes comprising HEK293E cells every other day (starting at day 0) to increase the final concentration from 1 mmol/l to 4 mmol/l in 1 mmol/l increments. Similarly, valproic acid was added to the corresponding tubes comprising CHO DG44 cells every other day (starting at day 0) to increase the final concentration from 0.125 mmol/l to 0.5 mmol/l in 0.125 mmol/l increments. The controls received 4 mmol/l valproic acid final concentration at day 0 (HEK293E) and 0.5 mmol/l (CHO DG44), respectively. The cells were incubated in a Kuhner shaker at 37° C. in a 5% CO₂ atmosphere under shaking at 180 rpm. 10 days post transfectionem, 200 μl of supernatant was removed from the cells in order to determine the antibody titer via ELISA.

[0111]The ELISA was performed as published in prior art [3]. In short, Goat anti-human kappa light chain IgG (Biosource) was used for coating the ELISA-plates, and with AP-conjugated goat anti-human gamma chain IgG (Biosource) the synthesized IgG1 was detected. NPP was used as a substrate for the alkaline phosphatase. Absorption was measured at 405 nm against 490 nm using a microplate reader (SPECTRAmax®340; Molecular Devices, Palo Alto, Calif., USA).

[0112]The following results were obtained at day 10 after transfection for HEK293E cells:

TABLE-US-00005 Standard Final concentration Batch titer (mg/l) deviation (mg/l) Control (4 mmol/l VPA at day 0) 193 21 VPA (escalating dosage) 182 20

[0113]As one can see, batch titers are statistically not different if one adds VPA directly at the beginning or slowly increases the concentration over time. Yet, given that--from a process point of view--it will be easier to add VPA only once, direct addition of VPA to the final concentration should be preferred.

[0114]The following results were obtained at day 10 after transfection for CHO cells:

TABLE-US-00006 Final concentration Batch titer (mg/l) Standard deviation (mg/l) Control (0.5 mmol/l VPA at 35.8 4.2 day 0) VPA (escalating dosage) 37.9 3.1

[0115]As one can see, batch titers are statistically not different if one adds VPA directly at the beginning or slowly increases the concentration over time. Yet, given that--from a process point of view--it will be easier to add VPA only once, direct addition of VPA to the final concentration should be preferred.

EXAMPLE 4

Alternative Transgenes

[0116]10 million suspension-adapted HEK293E cells [1] were resuspended in 0.5 ml of Ex-Cell 293 HEK 293 serum-free medium with 4 mmol/l L-glutamine (Cat. No. 14571C-1000M; Lot No. 6A0093; SAFC Biosciences, Lenexa, Kans., USA, "Ex-Cell" medium; note: "Ex-Cell" medium--as used herein--refers to a medium comprising 4 mmol/l L-glutamine) in a 50-ml filter tube each (TPP AG, Trasadingen, Switzerland; Cat. No. 87050, Lot 20050174). In a separate set, 10 million suspension-adapted CHO DG44 cells [6] were resuspended in 0.5 ml of ProCHO5 serum-free medium with 4 mmol/l L-glutamine and 0.68 g/l hypoxanthine and 0.194 g/l thymidine (BioWhittaker (Lonza), Belgium; Cat. No. BE12-766Q; Lot No. 7MB0040; "ProCHO5" medium; note: "ProCHO5" medium--as used herein--refers to a medium comprising 4 mmol/l L-glutamine and 0.68 g/l hypoxanthine and 0.194 g/l thymidine) in a 50-ml filter tube each (TPP AG, Trasadingen, Switzerland; Cat. No. 87050, Lot 20050174).

[0117]Then 12.5 μg plasmid DNA at a concentration of 1 μg/μl was added in each tube with the following composition

[0118]100% (12.5 μg) p-TNFR-Fc (SEQ ID NO: 11).

[0119]Plasmid p-TNFR-Fc comprises the genetic information for the production of an Fc-tagged, soluble TNFα receptor. The particular vector construct used in the present invention can also be obtained from the inventors. The inventors can be contacted via e-mail at hildinger@gmx.net. Moreover, commercial services do exist that produce any desired nucleotide sequence even comprising several kilobase pairs including complete expression cassettes in plasmid backgrounds (e.g., Invitrogen, Carlsbad, USA; Geneart, Germany.) Thus, providing the genetic sequence information of the plasmids should enable one of ordinary skill in the art to order the plasmids of the present invention at one of the commercial services listed.

[0120]After shortly mixing the DNA with the cells by gentle shaking, 25 μg of 25-kd linear polyethyleneimine ("PEI"; Polysciences, Eppelheim, Germany; [2]) were added at a concentration of 1 μg/μl to each of the tubes. After shortly mixing by gentle shaking, the filter tubes containing the cells together with the DNA and PEI were transferred into an orbital shaker (Kuhner Shaker Cabinet ISF-4-W, "Kuhner shaker", Kuhner AG, Birsfelden, Switzerland), and the cells were incubated for 4 hours at 37° C. in a 5% CO₂ atmosphere under shaking at 180 rpm. After that time, 4.5 ml of Ex-Cell medium were added to each of the filter tubes containing HEK 293 cells and 4.5 ml of Pro-CHO5 were added to each of the filter tubes containing CHO DG44 cells. Then, valproic acid was added to each of the tubes at the corresponding final concentration. The cells then were returned into the Kuhner shaker and incubated at 37° C. in a 5% CO₂ atmosphere under shaking at 180 rpm. Five days post transfectionem, 200 μl of supernatant was removed from the cells in order to determine the antibody titer via ELISA.

[0121]The ELISA was performed as published in prior art [3], but with a different coating antibody. In short, Goat Anti-Human IgG (Fc Fragment specific; Jackson ImmunoResearch Laboratories Inc., West Grove, Pa., USA; Code Number 109-006-098; Lot No. 72349) was used for coating the ELISA-plates, and with AP-conjugated goat anti-human gamma chain IgG (Biosource) the synthesized TNFR-Fc was detected. NPP was used as a substrate for the alkaline phosphatase. Absorption was measured at 405 nm against 490 nm using a microplate reader (SPECTRAmax®340; Molecular Devices, Palo Alto, Calif., USA).

[0122]The following results were obtained at day 5 after transfection for HEK293E cells with standard deviation given as +/-values:

TABLE-US-00007 Final VPA concentration Batch titer (mg/l) Fold increase over control 0 mmol/l (control) 35.5 ± 2.8 1 3.8 mmol/l 159.5 ± 12.0 4.5

[0123]As one can see, batch titers are increasing when adding valproic acid when using other (non-IgG) transgenes.

[0124]The following results were obtained at day 5 after transfection for CHO cells with standard deviation given as +/-values:

TABLE-US-00008 Final VPA concentration Batch titer (mg/l) Fold increase over control 0 mmol/l (control) 15.4 ± 1.3 1 0.5 mmol/l 27.7 ± 3.7 1.8

[0125]As one can see, batch titers are increasing when adding valproic acid when using other (non-IgG) transgenes.

EXAMPLE 5

Stable Cell Lines

[0126]20 million suspension-adapted HEK293E cells [1] were resuspended in 1 ml of Ex-Cell 293 HEK 293 serum-free medium with 4 mmol/l L-glutamine (Cat. No. 14571C-1000M; Lot No. 6A0093; SAFC Biosciences, Lenexa, Kans., USA, "Ex-Cell" medium; note: "Ex-Cell" medium--as used herein--refers to a medium comprising 4 mmol/l L-glutamine) in a 50-ml filter tube each (TPP AG, Trasadingen, Switzerland; Cat. No. 87050, Lot 20050174).

[0127]Then 50 μg plasmid DNA at a concentration of 1 μg/μl was added in each tube with the following composition

[0128]5% (2.5 μg) p-LC (SEQ ID NO: 1)

[0129]5% (2.5 μg) p-HC (SEQ ID NO: 3)

[0130]90% (45 μg) p-puro (SEQ ID NO: 13).

[0131]Plasmid p-LC comprises the genetic information for the production of the light chain of an IgG antibody, plasmid p-HC comprises the genetic information for the production of the heavy chain of an IgG antibody, plasmid p-puro comprises the genetic information for the enzyme puromycin-N-acetyl-transferase, which is encoded by the pac gene, the puromycin resistance gene. Expression of puromycin-N-acetyl-transferase confers resistance towards the antibiotic puromycin. The particular vector constructs used in the present invention can also be obtained from the inventors. The inventors can be contacted via e-mail at hildinger@gmx.net. Moreover, commercial services do exist that produce any desired nucleotide sequence even comprising several kilobase pairs including complete expression cassettes in plasmid backgrounds (e.g., Invitrogen, Carlsbad, USA; Geneart, Germany.) Thus, providing the genetic sequence information of the plasmids should enable one of ordinary skill in the art to order the plasmids of the present invention at one of the commercial services listed.

[0132]After shortly mixing the DNA with the cells by gentle shaking, 100 μg of 25-kd linear polyethyleneimine ("PEI"; Polysciences, Eppelheim, Germany [2]) were added at a concentration of 1 μg/μl to each of the tubes. After shortly mixing by gentle shaking, the filter tubes containing the cells together with the DNA and PEI were transferred into an orbital shaker (Kuhner Shaker Cabinet ISF-4-W, "Kuhner shaker", Kuhner AG, Birsfelden, Switzerland), and the cells were incubated for 3 hours at 37° C. in a 5% CO₂ atmosphere under shaking at 180 rpm. After that time, 4 ml of Ex-Cell medium were added to each of the filter tubes. One day after transfection, puromycin was added to a final concentration of 10 μg/ml (at this concentration of puromycin, only cells expressing the puromycin resistance gene can survive). The cells then were returned into the Kuhner shaker and incubated at 37° C. in a 5% CO₂ atmosphere under shaking at 180 rpm.

[0133]One month after transfection, the surviving cells were pooled and tested for IgG expression to verify successful genomic integration of p-LC and p-HC by Southern blotting and PCR (data not shown). The stable mass culture was then split into two tube spins (10 ml each), and VPA was added to a final concentration of 4 mmol/l in one of the two tubes.

[0134]The same experiment was performed with CHO cells with the exception of using ProCHO5 as a medium and adding VPA to a final concentration of 0.5 mmol/l in one of the two tubes.

[0135]5 days after VPA addition, 200 μl of supernatant was removed from the cells in order to determine the antibody titer via ELISA.

[0136]The ELISA was performed as published in prior art [3]. In short, Goat anti-human kappa light chain IgG (Biosource) was used for coating the ELISA-plates, and with AP-conjugated goat anti-human gamma chain IgG (Biosource) the synthesized IgG1 was detected. NPP was used as a substrate for the alkaline phosphatase. Absorption was measured at 405 nm against 490 nm using a microplate reader (SPECTRAmax®340; Molecular Devices, Palo Alto, Calif., USA).

[0137]The following results were obtained at day 5 with standard deviation given as +/-values:

TABLE-US-00009 Batch titer Final VPA concentration HEK293E (mg/l) Batch titer CHO (mg/l) 0 mmol/l (control) 9.5 ± 1.1 12.8 ± 1.4 0.5 mmol/l n/a 35.5 ± 2.5 4 mmol/l 47.8 ± 4.8 n/a

As one can see, VPA also increases batch titer for mammalian cells with stably integrated expression cassettes.

EXAMPLE 6

Combination with IDMTs

[0138]10 million suspension-adapted HEK293E cells [1] were resuspended in 0.5 ml of Ex-Cell 293 HEK 293 serum-free medium with 4 mmol/l L-glutamine (Cat. No. 14571C-1000M; Lot No. 6A0093; SAFC Biosciences, Lenexa, Kans., USA, "Ex-Cell" medium; note: "Ex-Cell" medium--as used herein--refers to a medium comprising 4 mmol/l L-glutamine) in a 50-ml filter tube each (TPP AG, Trasadingen, Switzerland; Cat. No. 87050, Lot 20050174). In a separate set, 10 million suspension-adapted CHO DG44 cells [6] were resuspended in 0.5 ml of ProCHO5 serum-free medium with 4 mmol/l L-glutamine and 0.68 g/l hypoxanthine and 0.194 g/l thymidine (BioWhittaker (Lonza), Belgium; Cat. No. BE12-766Q; Lot No. 7MB0040; "ProCHO5" medium; note: "ProCHO5" medium--as used herein--refers to a medium comprising 4 mmol/l L-glutamine and 0.68 g/l hypoxanthine and 0.194 g/l thymidine) in a 50-ml filter tube each (TPP AG, Trasadingen, Switzerland; Cat. No. 87050, Lot 20050174).

[0139]Then 12.5 μg plasmid DNA at a concentration of 1 μg/μl was added in each tube with the following composition

[0140]50% (6.25 μg) p-LC (SEQ ID NO: 1)

[0141]50% (6.25 μg) p-HC (SEQ ID NO: 3).

[0142]Plasmid p-LC comprises the genetic information for the production of the light chain of an IgG antibody, plasmid p-HC comprises the genetic information for the production of the heavy chain of an IgG antibody. The particular vector constructs used in the present invention can also be obtained from the inventors. The inventors can be contacted via e-mail at hildinger@gmx.net. Moreover, commercial services do exist that produce any desired nucleotide sequence even comprising several kilobase pairs including complete expression cassettes in plasmid backgrounds (e.g., Invitrogen, Carlsbad, USA; Geneart, Germany.) Thus, providing the genetic sequence information of the plasmids should enable one of ordinary skill in the art to order the plasmids of the present invention at one of the commercial services listed.

[0143]After shortly mixing the DNA with the cells by gentle shaking, 25 μg of 25-kd linear polyethyleneimine ("PEI"; Polysciences, Eppelheim, Germany; [2]) were added at a concentration of 1 μg/μl to each of the tubes. After shortly mixing by gentle shaking, the filter tubes containing the cells together with the DNA and PEI were transferred into an orbital shaker (Kuhner Shaker Cabinet ISF-4-W, "Kuhner shaker", Kuhner AG, Birsfelden, Switzerland), and the cells were incubated for 4 hours at 37° C. in a 5% CO₂ atmosphere under shaking at 180 rpm. After that time, 4.5 ml of Ex-Cell medium were added to each of the filter tubes containing HEK 293 cells and 4.5 ml of Pro-CHO5 were added to each of the filter tubes containing CHO DG44 cells. Then, valproic acid was added to each of the tubes at the corresponding final concentration. In addition, the following chemicals were added to the corresponding tubes at a final concentration of:

TABLE-US-00010 Chemical added (final concentration) HEK-293E cells CHO cells VPA 4 mmol/l 0.5 mmol/l Azacytidine 6 μmol/l 4 μmol/l RG108 8 μmol/l 60 μmol/l

[0144]Azacytidine was purchased from Sigma-Aldrich Chemie GmbH, Industriestrasse 25, CH-9471 Buchs SG, Catalog Number A1287-1 VL. RG108 was purchased from Sigma-Aldrich Chemie GmbH, Industriestrasse 25, CH-9471 Buchs SG, Catalog Number R8279-10MG.

[0145]The cells then were returned into the Kuhner shaker and incubated at 37° C. in a 5% CO₂ atmosphere under shaking at 180 rpm. Five days post transfectionem, 200 μl of supernatant was removed from the cells in order to determine the antibody titer via ELISA.

[0146]The ELISA was performed as published in prior art [3]. In short, Goat anti-human kappa light chain IgG (Biosource) was used for coating the ELISA-plates, and with AP-conjugated goat anti-human gamma chain IgG (Biosource) the synthesized IgG1 was detected. NPP was used as a substrate for the alkaline phosphatase. Absorption was measured at 405 nm against 490 nm using a microplate reader (SPECTRAmax®340; Molecular Devices, Palo Alto, Calif., USA).

[0147]The following results were obtained at day 5 after transfection for HEK293E cells with standard deviation given as +/-values:

TABLE-US-00011 Enhancer addition Fold increase in batch yield (final concentration) over control No addition 100% VPA 520% ± 30% VPA + Azacytidine 510% ± 35% VPA + RG108 525% ± 30% Azacytidine 160% ± 15% RG108 150% ± 20%

[0148]The following results were obtained at day 5 after transfection for CHO cells with standard deviation given as +/-values:

TABLE-US-00012 Enhancer addition Fold increase in batch yield (final concentration) over control No addition 100% VPA 170% ± 20% VPA + Azacytidine 350% ± 40% VPA + RG108 340% ± 35% Azacytidine 160% ± 10% RG108 170% ± 15%

[0149]As one can see, Azacytidine and RG108 act synergistically together with VPA in increasing batch yield in the context of CHO cells.

PREFERRED EMBODIMENT

[0150]20 million suspension-adapted HEK293E cells [1] were resuspended in 1 ml of Ex-Cell 293 HEK 293 serum-free medium with 4 mmol/l L-glutamine (Cat. No. 14571C-1000M; Lot No. 6A0093; SAFC Biosciences, Lenexa, Kans., USA, "Ex-Cell" medium; note: "Ex-Cell" medium--as used herein--refers to a medium comprising 4 mmol/l L-glutamine) in a 50-ml filter tube each (TPP AG, Trasadingen, Switzerland; Cat. No. 87050, Lot 20050174).

[0151]Then 50 μg plasmid DNA at a concentration of 1 μg/μl was added in each tube with the following composition

[0152]40% (20 μg) p-LC (SEQ ID NO: 1)

[0153]40% (20 μg) p-HC (SEQ ID NO: 3)

[0154]10% (5 μg) p-p18 (SEQ ID NO: 5)

[0155]10% (5 μg) p-p21 (SEQ ID NO: 7).

[0156]Plasmid p-LC comprises the genetic information for the production of the light chain of an IgG antibody, plasmid p-HC comprises the genetic information for the production of the heavy chain of an IgG antibody, plasmid p-p18 comprises the genetic information for the protein p18, and plasmid p-p21 comprises the genetic information for the protein p21. The particular vector constructs used in the present invention can also be obtained from the inventors. The inventors can be contacted via e-mail at hildinger@gmx.net. Moreover, commercial services do exist that produce any desired nucleotide sequence even comprising several kilobase pairs including complete expression cassettes in plasmid backgrounds (e.g., Invitrogen, Carlsbad, USA; Geneart, Germany.) Thus, providing the genetic sequence information of the plasmids should enable one of ordinary skill in the art to order the plasmids of the present invention at one of the commercial services listed.

[0157]After shortly mixing the DNA with the cells by gentle shaking, 100 μg of 25-kd linear polyethyleneimine ("PEI"; Polysciences, Eppelheim, Germany; [2]) were added at a concentration of 1 μg/μl to each of the tubes. After shortly mixing by gentle shaking, the filter tubes containing the cells together with the DNA and PEI were transferred into an orbital shaker (Kuhner Shaker Cabinet ISF-4-W, "Kuhner shaker", Kuhner AG, Birsfelden, Switzerland), and the cells were incubated for 3 hours at 37° C. in a 5% CO₂ atmosphere under shaking at 180 rpm. After that time, 4 ml of Ex-Cell medium were added to each of the filter tubes. Then, valproic acid or sodium butyrate (Cat. No. B5887-1G; Sigma-Aldrich Chemie GmbH, Industriestrasse 25, CH-9471 Buchs SG) was added to the corresponding tubes at the corresponding final concentrations. The cells then were returned into the Kuhner shaker and incubated at 37° C. in a 5% CO₂ atmosphere under shaking at 180 rpm. 14 days post transfectionem, 200 μl of supernatant was removed from the cells in order to determine the antibody titer via ELISA.

[0158]The ELISA was performed as published in prior art [3]. In short, Goat anti-human kappa light chain IgG (Biosource) was used for coating the ELISA-plates, and with AP-conjugated goat anti-human gamma chain IgG (Biosource) the synthesized IgG1 was detected. NPP was used as a substrate for the alkaline phosphatase. Absorption was measured at 405 nm against 490 nm using a microplate reader (SPECTRAmax®340; Molecular Devices, Palo Alto, Calif., USA).

[0159]The following results were obtained at day 14 after transfection for HEK293E cells with standard deviation given as +/-values:

TABLE-US-00013 Fold increase Enhancing agent Batch titer (mg/l) over control None 73 ± 6.5 1 3.8 mmol/l VPA 962 ± 73 13.2 3 mmol/l sodium butyrate 528 ± 42 7.2

[0160]As one can see, batch titers are higher when adding VPA compared to adding sodium butyrate. (3 mmol/l sodium butyrate was determined to be the optimal concentration in combination with HEK293E cells). Based on the smaller difference between sodium butyrate and VPA at day 5 (see example 2), it seems that VPA exerts its effect at least in part by preserving viability/cellular productivity for a longer period of time.

PRIOR ART CITATIONS

[0161]1. DUROCHER, Y, et al., A reporter gene assay for high-throughput screening of G-protein-coupled receptors stably or transiently expressed in HEK293 EBNA cells grown in suspension culture. Anal Biochem, 2000. 284(2): p. 316-26. [0162]2. BALDI, L, et al., Transient gene expression in suspension HEK-293 cells: application to large-scale protein production. Biotechnol Prog, 2005. 21(1): p. 148-53. [0163]3. MEISSNER, P, et al., Transient gene expression: recombinant protein production with suspension-adapted HEK293-EBNA cells. Biotechnol Bioeng, 2001. 75(2): p. 197-203. [0164]4. STETTLER, M, et al., New disposable tubes for rapid and precise biomass assessment for suspension cultures of mammalian cells. Biotechnol Bioeng, 2006. [0165]5. FUSSENEGGER, M, et al., Controlled proliferation by multigene metabolic engineering enhances the productivity of Chinese hamster ovary cells. Nat Biotechnol, 1998. 16(5): p. 468-72. [0166]6. URLAUB, G, et al., Deletion of the diploid dihydrofolate reductase locus from cultured mammalian cells. Cell, 1983. 33(2):405-12 [0167]7. GALBRAITH, D. J., et al., Control of Culture Environment for Improved Polyethylenimine-Mediated Transient Production of Recombinant Monoclonal Antibodies by CHO Cells. Biotechnol. Prog. 2006, 22, 753-762 [0168]Some relevant prior art U.S. Pat. Nos. 5,681,718, 6,117,652, 6,740,505

Sequence CWU 1

1415518DNAArtificialrecombinant plasmid p-LC 1agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 60acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 120tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 180ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccagattta 240attaaggctg cgcgctcgct cgctcactga ggccgcccgg gcaaagcccg ggcgtcgggc 300gacctttggt cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc 360catcactagg ggttccttgt agttaatgat taacccgcca tgctacttat ctacgtagcc 420atgctctagg aagatcggaa ttcgccctta agctagagtc gacaaagcta gctagttatt 480aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat 540aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa 600taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg 660agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc 720cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct 780tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga 840tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa 900gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc 960caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg 1020aggtctatat aagcagagct ggtttagtga accgtcagat cctgcagaag ttggtcgtga 1080ggcactgggc aggtaagtat caaggttaca agacaggttt aaggagacca atagaaactg 1140ggcttgtcga gacagagaag actcttgcgt ttctgatagg cacctattgg tcttactgac 1200atccactttg cctttctctc cacaggtgtc caggcggccg cct atg agt gtg ctc 1255 Met Ser Val Leu 1act cag gtc ctg gcg ttg ctg ctg ctg tgg ctt aca ggt acg cgt tgt 1303Thr Gln Val Leu Ala Leu Leu Leu Leu Trp Leu Thr Gly Thr Arg Cys5 10 15 20gac atc gtg atg acc cag tct cca tcc tcc ctg tct gca tct gta gga 1351Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 25 30 35gac aga gtc acc atc act tgc cgg gca agt cag aac att atc cgc tat 1399Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asn Ile Ile Arg Tyr 40 45 50tta aat tgg tat cag cag aag cca ggg aaa gcc cct agg ctc ctg atc 1447Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Arg Leu Leu Ile55 60 65tat ggt gcg tcc act ttg caa agt ggg gtc cca tca agg ttc agt ggc 1495Tyr Gly Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly70 75 80agt gga tct ggg aca gat ttc act ctc acc atc agt agt ctg caa cct 1543Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro85 90 95 100gaa gat ttt gca act tac tac tgt caa cag agt tac cgt acc cct cca 1591Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Arg Thr Pro Pro 105 110 115ttc act ttc ggc cct ggg acc aag ctt gaa atc aaa cct gtg gct gca 1639Phe Thr Phe Gly Pro Gly Thr Lys Leu Glu Ile Lys Pro Val Ala Ala 120 125 130cca tct gtc ttc atc ttc ccg cca tct gat gag cag ttg aaa tct gga 1687Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly135 140 145act gcc tct gtt gtg tgc ctg ctg aat aac ttc tat ccc aga gag gcc 1735Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala150 155 160aaa gta cag tgg aag gtg gat aac gcc ctc caa tcg ggt aac tcc cag 1783Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln165 170 175 180gag agt gtc aca gag cag gac agc aag gac agc acc tac agc ctc agc 1831Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 185 190 195agc acc ctg acg ctg agc aaa gca gac tac gag aaa cac aaa gtc tac 1879Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 200 205 210gcc tgc gaa gtc acc cat cag ggc ctg agc tcg ccc gtc aca aag agc 1927Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser215 220 225ttc aac agg gga gag tgt tag aagcttggat ccaatcaacc tctggattac 1978Phe Asn Arg Gly Glu Cys230aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga 2038tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc 2098tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa 2158cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc 2218acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc 2278atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc 2338gtggtgttgt cggggaagct gacgtccttt ccatggctgc tcgcctgtgt tgccacctgg 2398attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct 2458tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgagatct gcctcgactg 2518tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg 2578aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga 2638gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg 2698aagacaatag caggcatgct ggggactcga gattctagag tcgagttaag ggcgaattcc 2758cgattaggat cttcctagag catggctacg tagataagta gcatggcggg ttaatcatta 2818actacaagga acccctagtg atggagttgg ccactccctc tctgcgcgct cgctcgctca 2878ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg gcctcagtga 2938gcgagcgagc gcgcagcctt aattaaccta attcactggc cgtcgtttta caacgtcgtg 2998actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca 3058gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga 3118atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc 3178gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt 3238cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag 3298ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt 3358cacgtagtgg gccatcgccc cgatagacgg tttttcgccc tttgacgctg gagttcacgt 3418tcctcaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt 3478cttttgattt ataagggatt tttccgattt cggcctattg gttaaaaaat gagctgattt 3538aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tataatttca ggtggcatct 3598ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt 3658atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta 3718tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg 3778tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac 3838gagtgggtta catcgaactg gatctcaata gtggtaagat ccttgagagt tttcgccccg 3898aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc 3958gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg 4018ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat 4078gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg 4138gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg 4198atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc 4258ctgtagtaat ggtaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt 4318cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct 4378cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc 4438gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca 4498cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct 4558cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt 4618taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga 4678ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca 4738aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac 4798caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg 4858taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag 4918gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac 4978cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt 5038taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg 5098agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc 5158ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc 5218gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc 5278acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa 5338acgccagcaa cgcggccttt ttacggttcc tggccttttg ctgcggtttt gctcacatgt 5398tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg 5458ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag 55182234PRTArtificialSynthetic Construct 2Met Ser Val Leu Thr Gln Val Leu Ala Leu Leu Leu Leu Trp Leu Thr1 5 10 15Gly Thr Arg Cys Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser 20 25 30Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asn35 40 45Ile Ile Arg Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro50 55 60Arg Leu Leu Ile Tyr Gly Ala Ser Thr Leu Gln Ser Gly Val Pro Ser65 70 75 80Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 85 90 95Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr 100 105 110Arg Thr Pro Pro Phe Thr Phe Gly Pro Gly Thr Lys Leu Glu Ile Lys115 120 125Pro Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln130 135 140Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr145 150 155 160Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 165 170 175Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 180 185 190Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys195 200 205His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro210 215 220Val Thr Lys Ser Phe Asn Arg Gly Glu Cys225 23036238DNAArtificialrecombinant plasmid p-HC 3agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 60acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 120tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 180ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccagattta 240attaaggctg cgcgctcgct cgctcactga ggccgcccgg gcaaagcccg ggcgtcgggc 300gacctttggt cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc 360catcactagg ggttccttgt agttaatgat taacccgcca tgctacttat ctacgtagcc 420atgctctagg aagatcggaa ttcgccctta agctagagtc gacaaagcta gctagttatt 480aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat 540aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa 600taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg 660agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc 720cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct 780tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga 840tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa 900gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc 960caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg 1020aggtctatat aagcagagct ggtttagtga accgtcagat cctgcagaag ttggtcgtga 1080ggcactgggc aggtaagtat caaggttaca agacaggttt aaggagacca atagaaactg 1140ggcttgtcga gacagagaag actcttgcgt ttctgatagg cacctattgg tcttactgac 1200atccactttg cctttctctc cacaggtgtc caggcggccg cc atg gct tgg gtg 1254 Met Ala Trp Val 1tgg acc ttg cca ttc ctg atg gca gct gcc caa ggt gtc gac gca cag 1302Trp Thr Leu Pro Phe Leu Met Ala Ala Ala Gln Gly Val Asp Ala Gln5 10 15 20gtg aaa ctg ctc gag tct ggg gga ggc gtg gtc cag ccg ggg ggg tcc 1350Val Lys Leu Leu Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly Ser 25 30 35ctg aga ctc tcc tgt gaa gcg tct gga ttc gcc ctc aga agt tct ggc 1398Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Ala Leu Arg Ser Ser Gly 40 45 50atg cac tgg gtc cgc cag gct cct ggc aag ggg ctg gag tgg gtg gca 1446Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala55 60 65ctt ata tgg ttt gat gga agt atc aga tcg tat gca gaa tcc gtg aag 1494Leu Ile Trp Phe Asp Gly Ser Ile Arg Ser Tyr Ala Glu Ser Val Lys70 75 80ggc cga ttc acc atc tcc aga gac act tcc aag aac acc cta tat ctc 1542Gly Arg Phe Thr Ile Ser Arg Asp Thr Ser Lys Asn Thr Leu Tyr Leu85 90 95 100caa atg cgc agt ctg agt gcc gac gac acg gct gtg tat tac tgt gcg 1590Gln Met Arg Ser Leu Ser Ala Asp Asp Thr Ala Val Tyr Tyr Cys Ala 105 110 115aga gac aag gcg gtt cgg gga att agc agg tac aac tat tac atg gac 1638Arg Asp Lys Ala Val Arg Gly Ile Ser Arg Tyr Asn Tyr Tyr Met Asp 120 125 130gtc tgg ggc aaa ggg acc acg gtc acc gtc tcc tca gcc tcc acc aag 1686Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys135 140 145ggc cca tcg gtc ttc ccc ctg gca ccc tcc tcc aag agc acc tct ggg 1734Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly150 155 160ggc aca gcg gcc ctg ggc tgc ctg gtc aag gac tac ttc ccc gaa ccg 1782Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro165 170 175 180gtg acg gtg tcg tgg aac tca ggc gcc ctg acc agc ggc gtg cac acc 1830Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 185 190 195ttc ccg gct gtc cta cag tcc tca gga ctc tac tcc ctc agc agc gtg 1878Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 200 205 210gtg acc gtg ccc tcc agc agc ttg ggc acc cag acc tac atc tgc aac 1926Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn215 220 225gtg aat cac aag ccc agc aac acc aag gtg gac aag aga gtt gag ccc 1974Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro230 235 240aaa tct tgt gac aaa act cac aca tgc cca ccg tgc cca gca cct gaa 2022Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu245 250 255 260ctc ctg ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac 2070Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 265 270 275acc ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac 2118Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 280 285 290gtg agc cac gaa gac cct gag gtc aag ttc aac tgg tac gtg gac ggc 2166Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly295 300 305gtg gag gtg cat aat gcc aag aca aag ccg cgg gag gag cag tac aac 2214Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn310 315 320agc acg tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg 2262Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp325 330 335 340ctg aat ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca 2310Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 345 350 355gcc ccc atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa 2358Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 360 365 370cca cag gtg tac acc ctg ccc cca tcc cgg gag gag atg acc aag aac 2406Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn375 380 385cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc gac atc 2454Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile390 395 400gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac aag acc 2502Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr405 410 415 420acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tat agc aag 2550Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 425 430 435ctc acc gtg gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc 2598Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 440 445 450tcc gtg atg cat gag gct ctg cac aac cac tac acg cag aag agc ctc 2646Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu455 460 465tcc ctg tcc ccg ggt aaa tga taagcttgga tccaatcaac ctctggatta 2697Ser Leu Ser Pro Gly Lys470caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg 2757atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc 2817ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca 2877acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac 2937cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact 2997catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc 3057cgtggtgttg tcggggaagc tgacgtcctt tccatggctg ctcgcctgtg ttgccacctg 3117gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc 3177ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgagatc tgcctcgact 3237gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg 3297gaaggtgcca ctcccactgt cctttcctaa

taaaatgagg aaattgcatc gcattgtctg 3357agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg 3417gaagacaata gcaggcatgc tggggactcg agattctaga gtcgagttaa gggcgaattc 3477ccgattagga tcttcctaga gcatggctac gtagataagt agcatggcgg gttaatcatt 3537aactacaagg aacccctagt gatggagttg gccactccct ctctgcgcgc tcgctcgctc 3597actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg 3657agcgagcgag cgcgcagcct taattaacct aattcactgg ccgtcgtttt acaacgtcgt 3717gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc 3777agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg 3837aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg 3897cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct 3957tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta 4017gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt 4077tcacgtagtg ggccatcgcc ccgatagacg gtttttcgcc ctttgacgct ggagttcacg 4137ttcctcaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat 4197tcttttgatt tataagggat ttttccgatt tcggcctatt ggttaaaaaa tgagctgatt 4257taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttataatttc aggtggcatc 4317tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg 4377tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt 4437atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 4497gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca 4557cgagtgggtt acatcgaact ggatctcaat agtggtaaga tccttgagag ttttcgcccc 4617gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc 4677cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg 4737gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta 4797tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc 4857ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt 4917gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg 4977cctgtagtaa tggtaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct 5037tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc 5097tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct 5157cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac 5217acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc 5277tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat 5337ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg 5397accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc 5457aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa 5517ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag 5577gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta 5637ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta 5697ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag 5757ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg 5817gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg 5877cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag 5937cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc 5997cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa 6057aacgccagca acgcggcctt tttacggttc ctggcctttt gctgcggttt tgctcacatg 6117ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct 6177gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa 6237g 62384474PRTArtificialSynthetic Construct 4Met Ala Trp Val Trp Thr Leu Pro Phe Leu Met Ala Ala Ala Gln Gly1 5 10 15Val Asp Ala Gln Val Lys Leu Leu Glu Ser Gly Gly Gly Val Val Gln 20 25 30Pro Gly Gly Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Ala Leu35 40 45Arg Ser Ser Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu50 55 60Glu Trp Val Ala Leu Ile Trp Phe Asp Gly Ser Ile Arg Ser Tyr Ala65 70 75 80Glu Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Thr Ser Lys Asn 85 90 95Thr Leu Tyr Leu Gln Met Arg Ser Leu Ser Ala Asp Asp Thr Ala Val 100 105 110Tyr Tyr Cys Ala Arg Asp Lys Ala Val Arg Gly Ile Ser Arg Tyr Asn115 120 125Tyr Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser130 135 140Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys145 150 155 160Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 165 170 175Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 180 185 190Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser195 200 205Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr210 215 220Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys225 230 235 240Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 245 250 255Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 260 265 270Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys275 280 285Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp290 295 300Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu305 310 315 320Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 325 330 335His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 340 345 350Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly355 360 365Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu370 375 380Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr385 390 395 400Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 405 410 415Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 420 425 430Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn435 440 445Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr450 455 460Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys465 47055291DNAArtificialrecombinant plasmid p-p18 5agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 60acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 120tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 180ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccagattta 240attaaggctg cgcgctcgct cgctcactga ggccgcccgg gcaaagcccg ggcgtcgggc 300gacctttggt cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc 360catcactagg ggttccttgt agttaatgat taacccgcca tgctacttat ctacgtagcc 420atgctctagg aagatcggaa ttcgccctta agctagctag ttattaatag taatcaatta 480cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg 540gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc 600ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa 660ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca 720atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta 780cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt 840acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg 900acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca 960actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca 1020gagctggttt agtgaaccgt cagatcctgc agaagttggt cgtgaggcac tgggcaggta 1080agtatcaagg ttacaagaca ggtttaagga gaccaataga aactgggctt gtcgagacag 1140agaagactct tgcgtttctg ataggcacct attggtctta ctgacatcca ctttgccttt 1200ctctccacag gtgtccaggc ggccgcc atg gcc gag cct tgg ggg aac gag ttg 1254 Met Ala Glu Pro Trp Gly Asn Glu Leu 1 5gcg tcc gca gct gcc agg ggg gac cta gag caa ctt act agt ttg ttg 1302Ala Ser Ala Ala Ala Arg Gly Asp Leu Glu Gln Leu Thr Ser Leu Leu10 15 20 25caa aat aat gta aac gtc aat gca caa aat gga ttt gga agg act gcg 1350Gln Asn Asn Val Asn Val Asn Ala Gln Asn Gly Phe Gly Arg Thr Ala 30 35 40ctg cag gtt atg aaa ctt gga aat ccc gag att gcc agg aga ctg cta 1398Leu Gln Val Met Lys Leu Gly Asn Pro Glu Ile Ala Arg Arg Leu Leu 45 50 55ctt aga ggt gct aat ccc gat ttg aaa gac cga act ggt ttc gct gtc 1446Leu Arg Gly Ala Asn Pro Asp Leu Lys Asp Arg Thr Gly Phe Ala Val60 65 70att cat gat gcg gcc aga gca ggt ttc ctg gac act tta cag act ttg 1494Ile His Asp Ala Ala Arg Ala Gly Phe Leu Asp Thr Leu Gln Thr Leu75 80 85ctg gag ttt caa gct gat gtt aac atc gag gat aat gaa ggg aac ctg 1542Leu Glu Phe Gln Ala Asp Val Asn Ile Glu Asp Asn Glu Gly Asn Leu90 95 100 105ccc ttg cac ttg gct gcc aaa gaa ggc cac ctc cgg gtg gtg gag ttc 1590Pro Leu His Leu Ala Ala Lys Glu Gly His Leu Arg Val Val Glu Phe 110 115 120ctg gtg aag cac atg gcc agc aat gtg ggg cat cgg aac cat aag ggg 1638Leu Val Lys His Met Ala Ser Asn Val Gly His Arg Asn His Lys Gly 125 130 135gac acc gcc tgt gat ttg gcc agg ctc tat ggg agg aat gag gtt gtt 1686Asp Thr Ala Cys Asp Leu Ala Arg Leu Tyr Gly Arg Asn Glu Val Val140 145 150agc ctg atg cag gca aac ggg gct ggg gga gcc aca aat ctt caa taa 1734Ser Leu Met Gln Ala Asn Gly Ala Gly Gly Ala Thr Asn Leu Gln155 160 165taagcttgga tccaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct 1794taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc 1854tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct 1914ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga 1974cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc 2034tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac 2094aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaagc tgacgtcctt 2154tccatggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt 2214cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc 2274tcttccgcgt cttcgagatc tgcctcgact gtgccttcta gttgccagcc atctgttgtt 2334tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa 2394taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg 2454gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggactcg 2514agttaagggc gaattcccga ttaggatctt cctagagcat ggctacgtag ataagtagca 2574tggcgggtta atcattaact acaaggaacc cctagtgatg gagttggcca ctccctctct 2634gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc 2694ccgggcggcc tcagtgagcg agcgagcgcg cagccttaat taacctaatt cactggccgt 2754cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc 2814acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca 2874acagttgcgc agcctgaatg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc 2934gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc 2994tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa 3054tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact 3114tgattagggt gatggttcac gtagtgggcc atcgccccga tagacggttt ttcgcccttt 3174gacgctggag ttcacgttcc tcaatagtgg actcttgttc caaactggaa caacactcaa 3234ccctatctcg gtctattctt ttgatttata agggattttt ccgatttcgg cctattggtt 3294aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttat 3354aatttcaggt ggcatctttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta 3414aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata 3474ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc 3534ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga 3594agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaatagtg gtaagatcct 3654tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg 3714tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta 3774ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat 3834gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt 3894acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga 3954tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga 4014gcgtgacacc acgatgcctg tagtaatggt aacaacgttg cgcaaactat taactggcga 4074actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc 4134aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc 4194cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg 4254tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat 4314cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata 4374tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct 4434ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga 4494ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg 4554cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc 4614aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct 4674agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc 4734tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt 4794ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg 4854cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct 4914atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag 4974ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag 5034tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg 5094gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg 5154cggttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac 5214cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt 5274gagcgaggaa gcggaag 52916168PRTArtificialSynthetic Construct 6Met Ala Glu Pro Trp Gly Asn Glu Leu Ala Ser Ala Ala Ala Arg Gly1 5 10 15Asp Leu Glu Gln Leu Thr Ser Leu Leu Gln Asn Asn Val Asn Val Asn 20 25 30Ala Gln Asn Gly Phe Gly Arg Thr Ala Leu Gln Val Met Lys Leu Gly35 40 45Asn Pro Glu Ile Ala Arg Arg Leu Leu Leu Arg Gly Ala Asn Pro Asp50 55 60Leu Lys Asp Arg Thr Gly Phe Ala Val Ile His Asp Ala Ala Arg Ala65 70 75 80Gly Phe Leu Asp Thr Leu Gln Thr Leu Leu Glu Phe Gln Ala Asp Val 85 90 95Asn Ile Glu Asp Asn Glu Gly Asn Leu Pro Leu His Leu Ala Ala Lys 100 105 110Glu Gly His Leu Arg Val Val Glu Phe Leu Val Lys His Met Ala Ser115 120 125Asn Val Gly His Arg Asn His Lys Gly Asp Thr Ala Cys Asp Leu Ala130 135 140Arg Leu Tyr Gly Arg Asn Glu Val Val Ser Leu Met Gln Ala Asn Gly145 150 155 160Ala Gly Gly Ala Thr Asn Leu Gln 16575279DNAArtificialrecombinant plasmid p-p21 7agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 60acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 120tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 180ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccagattta 240attaaggctg cgcgctcgct cgctcactga ggccgcccgg gcaaagcccg ggcgtcgggc 300gacctttggt cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc 360catcactagg ggttccttgt agttaatgat taacccgcca tgctacttat ctacgtagcc 420atgctctagg aagatcggaa ttcgccctta agctagctag ttattaatag taatcaatta 480cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg 540gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc 600ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa 660ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca 720atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta 780cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt 840acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg 900acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca 960actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca 1020gagctggttt agtgaaccgt cagatcctgc agaagttggt cgtgaggcac tgggcaggta 1080agtatcaagg ttacaagaca ggtttaagga gaccaataga aactgggctt gtcgagacag 1140agaagactct tgcgtttctg ataggcacct attggtctta ctgacatcca ctttgccttt 1200ctctccacag gtgtccaggc ggccgcc atg tca gaa ccg gct ggg gat gtc cgt 1254 Met Ser Glu Pro Ala Gly Asp Val Arg 1 5cag aac cca tgc ggc agc aag gcc tgc cgc cgc ctc ttc ggc cca gtg 1302Gln Asn Pro Cys Gly Ser Lys Ala Cys Arg Arg Leu Phe Gly Pro Val10 15 20 25gac agc gag cag ctg aga cgc gac tgt gat gcg cta atg gcg ggc tgc 1350Asp Ser Glu Gln Leu Arg Arg Asp Cys Asp Ala Leu Met Ala Gly Cys 30 35 40atc cag gag gcc cgt gag cga tgg

aac ttc gac ttt gtc acc gag aca 1398Ile Gln Glu Ala Arg Glu Arg Trp Asn Phe Asp Phe Val Thr Glu Thr 45 50 55cca ctg gag ggt gac ttc gcc tgg gag cgt gtg cgg ggc ctt ggc ctg 1446Pro Leu Glu Gly Asp Phe Ala Trp Glu Arg Val Arg Gly Leu Gly Leu60 65 70ccc aag ctc tac ctt ccc acg ggg ccc cgg cga ggc cgg gat gag ttg 1494Pro Lys Leu Tyr Leu Pro Thr Gly Pro Arg Arg Gly Arg Asp Glu Leu75 80 85gga gga ggc agg cgg cct ggc acc tca cct gct ctg ctg cag ggg aca 1542Gly Gly Gly Arg Arg Pro Gly Thr Ser Pro Ala Leu Leu Gln Gly Thr90 95 100 105gca gag gaa gac cat gtg gac ctg tca ctg tct tgt acc ctt gtg cct 1590Ala Glu Glu Asp His Val Asp Leu Ser Leu Ser Cys Thr Leu Val Pro 110 115 120cgc tca ggg gag cag gct gaa ggg tcc cca ggt gga cct gga gac tct 1638Arg Ser Gly Glu Gln Ala Glu Gly Ser Pro Gly Gly Pro Gly Asp Ser 125 130 135cag ggt cga aaa cgg cgg cag acc agc atg aca gat ttc tac cac tcc 1686Gln Gly Arg Lys Arg Arg Gln Thr Ser Met Thr Asp Phe Tyr His Ser140 145 150aaa cgc cgg ctg atc ttc tcc aag agg aag ccc taa taagcttgga 1732Lys Arg Arg Leu Ile Phe Ser Lys Arg Lys Pro155 160tccaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt 1792gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc 1852cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag 1912ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc 1972actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc 2032cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg 2092ctgttgggca ctgacaattc cgtggtgttg tcggggaagc tgacgtcctt tccatggctg 2152ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc 2212ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt 2272cttcgagatc tgcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc 2332ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg 2392aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg 2452acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggactcg agttaagggc 2512gaattcccga ttaggatctt cctagagcat ggctacgtag ataagtagca tggcgggtta 2572atcattaact acaaggaacc cctagtgatg gagttggcca ctccctctct gcgcgctcgc 2632tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc 2692tcagtgagcg agcgagcgcg cagccttaat taacctaatt cactggccgt cgttttacaa 2752cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct 2812ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc 2872agcctgaatg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg 2932gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc 2992ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc 3052cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt 3112gatggttcac gtagtgggcc atcgccccga tagacggttt ttcgcccttt gacgctggag 3172ttcacgttcc tcaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg 3232gtctattctt ttgatttata agggattttt ccgatttcgg cctattggtt aaaaaatgag 3292ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttat aatttcaggt 3352ggcatctttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca 3412aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg 3472aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc 3532cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg 3592ggtgcacgag tgggttacat cgaactggat ctcaatagtg gtaagatcct tgagagtttt 3652cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta 3712ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat 3772gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga 3832gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca 3892acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact 3952cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc 4012acgatgcctg tagtaatggt aacaacgttg cgcaaactat taactggcga actacttact 4072ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt 4132ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt 4192gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt 4252atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata 4312ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag 4372attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat 4432ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa 4492aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca 4552aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt 4612ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct agtgtagccg 4672tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc 4732ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga 4792cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc 4852agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc 4912gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca 4972ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg 5032tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta 5092tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg cggttttgct 5152cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag 5212tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa 5272gcggaag 52798164PRTArtificialSynthetic Construct 8Met Ser Glu Pro Ala Gly Asp Val Arg Gln Asn Pro Cys Gly Ser Lys1 5 10 15Ala Cys Arg Arg Leu Phe Gly Pro Val Asp Ser Glu Gln Leu Arg Arg 20 25 30Asp Cys Asp Ala Leu Met Ala Gly Cys Ile Gln Glu Ala Arg Glu Arg35 40 45Trp Asn Phe Asp Phe Val Thr Glu Thr Pro Leu Glu Gly Asp Phe Ala50 55 60Trp Glu Arg Val Arg Gly Leu Gly Leu Pro Lys Leu Tyr Leu Pro Thr65 70 75 80Gly Pro Arg Arg Gly Arg Asp Glu Leu Gly Gly Gly Arg Arg Pro Gly 85 90 95Thr Ser Pro Ala Leu Leu Gln Gly Thr Ala Glu Glu Asp His Val Asp 100 105 110Leu Ser Leu Ser Cys Thr Leu Val Pro Arg Ser Gly Glu Gln Ala Glu115 120 125Gly Ser Pro Gly Gly Pro Gly Asp Ser Gln Gly Arg Lys Arg Arg Gln130 135 140Thr Ser Met Thr Asp Phe Tyr His Ser Lys Arg Arg Leu Ile Phe Ser145 150 155 160Lys Arg Lys Pro95252DNAArtificialrecombinant plasmid p-aFGF 9agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 60acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 120tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 180ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccagattta 240attaaggctg cgcgctcgct cgctcactga ggccgcccgg gcaaagcccg ggcgtcgggc 300gacctttggt cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc 360catcactagg ggttccttgt agttaatgat taacccgcca tgctacttat ctacgtagcc 420atgctctagg aagatcggaa ttcgccctta agctagctag ttattaatag taatcaatta 480cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg 540gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc 600ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa 660ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca 720atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta 780cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt 840acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg 900acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca 960actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca 1020gagctggttt agtgaaccgt cagatcctgc agaagttggt cgtgaggcac tgggcaggta 1080agtatcaagg ttacaagaca ggtttaagga gaccaataga aactgggctt gtcgagacag 1140agaagactct tgcgtttctg ataggcacct attggtctta ctgacatcca ctttgccttt 1200ctctccacag gtgtccaggc ggccgcc atg gct gaa ggg gaa atc acc acc ttc 1254 Met Ala Glu Gly Glu Ile Thr Thr Phe 1 5aca gcc ctg acc gag aag ttt aat ctg cct cca ggg aat tac aag aag 1302Thr Ala Leu Thr Glu Lys Phe Asn Leu Pro Pro Gly Asn Tyr Lys Lys10 15 20 25ccc aaa ctc ctc tac tgt agc aac ggg ggc cac ttc ctg agg atc ctt 1350Pro Lys Leu Leu Tyr Cys Ser Asn Gly Gly His Phe Leu Arg Ile Leu 30 35 40ccg gat ggc aca gtg gat ggg aca agg gac agg agc gac cag cac att 1398Pro Asp Gly Thr Val Asp Gly Thr Arg Asp Arg Ser Asp Gln His Ile 45 50 55cag ctg cag ctc agt gcg gaa agc gtg ggg gag gtg tat ata aag agt 1446Gln Leu Gln Leu Ser Ala Glu Ser Val Gly Glu Val Tyr Ile Lys Ser60 65 70acc gag act ggc cag tac ttg gcc atg gac acc gac ggg ctt tta tac 1494Thr Glu Thr Gly Gln Tyr Leu Ala Met Asp Thr Asp Gly Leu Leu Tyr75 80 85ggc tca cag aca cca aat gag gaa tgt ttg ttc ctg gaa agg ctg gag 1542Gly Ser Gln Thr Pro Asn Glu Glu Cys Leu Phe Leu Glu Arg Leu Glu90 95 100 105gag aac cat tac aac acc tat ata tcc aag aag cat gca gag aag aat 1590Glu Asn His Tyr Asn Thr Tyr Ile Ser Lys Lys His Ala Glu Lys Asn 110 115 120tgg ttt gtt ggc ctc aag aag aat ggg agc tgc aaa cgc ggt cct cgg 1638Trp Phe Val Gly Leu Lys Lys Asn Gly Ser Cys Lys Arg Gly Pro Arg 125 130 135act cac tat ggc cag aaa gca atc ttg ttt ctc ccc ctg cca gtc tct 1686Thr His Tyr Gly Gln Lys Ala Ile Leu Phe Leu Pro Leu Pro Val Ser140 145 150tct gat taa taagcttgga tccaatcaac ctctggatta caaaatttgt 1735Ser Asp155gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct 1795ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat 1855aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg 1915gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag 1975ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc 2035tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg 2095tcggggaagc tgacgtcctt tccatggctg ctcgcctgtg ttgccacctg gattctgcgc 2155gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc 2215ctgctgccgg ctctgcggcc tcttccgcgt cttcgagatc tgcctcgact gtgccttcta 2275gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca 2335ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc 2395attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata 2455gcaggcatgc tggggactcg agttaagggc gaattcccga ttaggatctt cctagagcat 2515ggctacgtag ataagtagca tggcgggtta atcattaact acaaggaacc cctagtgatg 2575gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc 2635gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg cagccttaat 2695taacctaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc 2755caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc 2815cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggga cgcgccctgt 2875agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc 2935agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc 2995tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg 3055cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccccga 3115tagacggttt ttcgcccttt gacgctggag ttcacgttcc tcaatagtgg actcttgttc 3175caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata agggattttt 3235ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt 3295aacaaaatat taacgtttat aatttcaggt ggcatctttc ggggaaatgt gcgcggaacc 3355cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc 3415tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc 3475gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg 3535gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat 3595ctcaatagtg gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc 3655acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa 3715ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa 3775aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt 3835gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct 3895tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat 3955gaagccatac caaacgacga gcgtgacacc acgatgcctg tagtaatggt aacaacgttg 4015cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg 4075atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt 4135attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg 4195ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg 4255gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg 4315tcagaccaag tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa 4375aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt 4435tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt 4495tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt 4555ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag 4615ataccaaata ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta 4675gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat 4735aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg 4795ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg 4855agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac 4915aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga 4975aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt 5035ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta 5095cggttcctgg ccttttgctg cggttttgct cacatgttct ttcctgcgtt atcccctgat 5155tctgtggata accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg 5215accgagcgca gcgagtcagt gagcgaggaa gcggaag 525210155PRTArtificialSynthetic Construct 10Met Ala Glu Gly Glu Ile Thr Thr Phe Thr Ala Leu Thr Glu Lys Phe1 5 10 15Asn Leu Pro Pro Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys Ser 20 25 30Asn Gly Gly His Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp Gly35 40 45Thr Arg Asp Arg Ser Asp Gln His Ile Gln Leu Gln Leu Ser Ala Glu50 55 60Ser Val Gly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Tyr Leu65 70 75 80Ala Met Asp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asn Glu 85 90 95Glu Cys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr Tyr 100 105 110Ile Ser Lys Lys His Ala Glu Lys Asn Trp Phe Val Gly Leu Lys Lys115 120 125Asn Gly Ser Cys Lys Arg Gly Pro Arg Thr His Tyr Gly Gln Lys Ala130 135 140Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp145 150 155116196DNAArtificialrecombinant plasmid p-Enbrel 11agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 60acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 120tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 180ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccagattta 240attaaggctg cgcgctcgct cgctcactga ggccgcccgg gcaaagcccg ggcgtcgggc 300gacctttggt cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc 360catcactagg ggttccttgt agttaatgat taacccgcca tgctacttat ctacgtagcc 420atgctctagg aagatcggaa ttcgccctta agctagagtc gacaaagcta gctagttatt 480aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat 540aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa 600taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg 660agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc 720cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct 780tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga 840tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa 900gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc 960caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg 1020aggtctatat aagcagagct ggtttagtga accgtcagat cctgcagaag ttggtcgtga 1080ggcactgggc aggtaagtat caaggttaca agacaggttt aaggagacca atagaaactg 1140ggcttgtcga gacagagaag actcttgcgt ttctgatagg cacctattgg tcttactgac 1200atccactttg cctttctctc cacaggtgtc caggcggccg cc atg gcg ccc gtc 1254 Met Ala Pro Val 1gcc gtc tgg gcc gcg ctg gcc gtc gga ctg gag ctc tgg gct gcg gcg 1302Ala Val Trp Ala Ala Leu Ala Val Gly Leu Glu Leu Trp Ala Ala Ala5 10 15 20cac gcc ttg ccc gcc cag gtg gca ttt aca ccc tac gcc ccg gag ccc 1350His Ala Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro 25 30 35ggg agc aca tgc cgg ctc aga gaa tac tat gac cag aca gct cag atg 1398Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met 40 45 50tgc tgc agc aaa tgc tcg ccg ggc caa cat gca aaa gtc ttc tgt acc 1446Cys Cys Ser Lys Cys Ser Pro Gly Gln His Ala Lys

Val Phe Cys Thr55 60 65aag acc tcg gac acc gtg tgt gac tcc tgt gag gac agc aca tac acc 1494Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr70 75 80cag ctc tgg aac tgg gtt ccc gag tgc ttg agc tgt ggc tcc cgc tgt 1542Gln Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys85 90 95 100agc tct gac cag gtg gaa act caa gcc tgc act cgg gaa cag aac cgc 1590Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg 105 110 115atc tgc acc tgc agg ccc ggc tgg tac tgc gcg ctg agc aag cag gag 1638Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu 120 125 130ggg tgc cgg ctg tgc gcg ccg ctg cgc aag tgc cgc ccg ggc ttc ggc 1686Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly135 140 145gtg gcc aga cca gga act gaa aca tca gac gtg gtg tgc aag ccc tgt 1734Val Ala Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys150 155 160gcc ccg ggg acg ttc tcc aac acg act tca tcc acg gat att tgc agg 1782Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg165 170 175 180ccc cac cag atc tgt aac gtg gtg gcc atc cct ggg aat gca agc atg 1830Pro His Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met 185 190 195gat gca gtc tgc acg tcc acg tcc ccc acc cgg agt atg gcc cca ggg 1878Asp Ala Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro Gly 200 205 210gca gta cac tta ccc cag cca gtg tcc aca cga tcc caa ctc gac gtt 1926Ala Val His Leu Pro Gln Pro Val Ser Thr Arg Ser Gln Leu Asp Val215 220 225gag ccc aaa tct tgt gac aaa act cac aca tgc cca ccg tgc cca gca 1974Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala230 235 240cct gaa ctc ctg ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc 2022Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro245 250 255 260aag gac acc ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg 2070Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 265 270 275gtg gac gtg agc cac gaa gac cct gag gtc aag ttc aac tgg tac gtg 2118Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 280 285 290gac ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg gag gag cag 2166Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln295 300 305tac aac agc acg tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag 2214Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln310 315 320gac tgg ctg aat ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc 2262Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala325 330 335 340ctc cca gcc ccc atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc 2310Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 345 350 355cga gaa cca cag gtg tac acc ctg ccc cca tcc cgg gag gag atg acc 2358Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 360 365 370aag aac cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc 2406Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser375 380 385gac atc gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac 2454Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr390 395 400aag acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tat 2502Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr405 410 415 420agc aag ctc acc gtg gac aag agc agg tgg cag cag ggg aac gtc ttc 2550Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 425 430 435tca tgc tcc gtg atg cat gag gct ctg cac aac cac tac acg cag aag 2598Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 440 445 450agc ctc tcc ctg tcc ccg ggt aaa tga taagcttgga tccaatcaac 2645Ser Leu Ser Leu Ser Pro Gly Lys455 460ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta 2705cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt 2765tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg 2825ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg 2885gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca 2945cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca 3005ctgacaattc cgtggtgttg tcggggaagc tgacgtcctt tccatggctg ctcgcctgtg 3065ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag 3125cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgagatc 3185tgcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc 3245cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc 3305gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg 3365ggaggattgg gaagacaata gcaggcatgc tggggactcg agattctaga gtcgagttaa 3425gggcgaattc ccgattagga tcttcctaga gcatggctac gtagataagt agcatggcgg 3485gttaatcatt aactacaagg aacccctagt gatggagttg gccactccct ctctgcgcgc 3545tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc 3605ggcctcagtg agcgagcgag cgcgcagcct taattaacct aattcactgg ccgtcgtttt 3665acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc 3725ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt 3785gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc gcattaagcg cggcgggtgt 3845ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc 3905tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg 3965gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta 4025gggtgatggt tcacgtagtg ggccatcgcc ccgatagacg gtttttcgcc ctttgacgct 4085ggagttcacg ttcctcaata gtggactctt gttccaaact ggaacaacac tcaaccctat 4145ctcggtctat tcttttgatt tataagggat ttttccgatt tcggcctatt ggttaaaaaa 4205tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttataatttc 4265aggtggcatc tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca 4325ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa 4385aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt 4445ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca 4505gttgggtgca cgagtgggtt acatcgaact ggatctcaat agtggtaaga tccttgagag 4565ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc 4625ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca 4685gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt 4745aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct 4805gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt 4865aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga 4925caccacgatg cctgtagtaa tggtaacaac gttgcgcaaa ctattaactg gcgaactact 4985tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc 5045acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga 5105gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt 5165agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga 5225gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact 5285ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga 5345taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt 5405agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca 5465aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct 5525ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta 5585gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct 5645aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc 5705aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca 5765gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga 5825aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg 5885aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt 5945cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag 6005cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctgcggttt 6065tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt 6125tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga 6185ggaagcggaa g 619612460PRTArtificialSynthetic Construct 12Met Ala Pro Val Ala Val Trp Ala Ala Leu Ala Val Gly Leu Glu Leu1 5 10 15Trp Ala Ala Ala His Ala Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr 20 25 30Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln35 40 45Thr Ala Gln Met Cys Cys Ser Lys Cys Ser Pro Gly Gln His Ala Lys50 55 60Val Phe Cys Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu Asp65 70 75 80Ser Thr Tyr Thr Gln Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys 85 90 95Gly Ser Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr Arg 100 105 110Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu115 120 125Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg130 135 140Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr Ser Asp Val Val145 150 155 160Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr 165 170 175Asp Ile Cys Arg Pro His Gln Ile Cys Asn Val Val Ala Ile Pro Gly 180 185 190Asn Ala Ser Met Asp Ala Val Cys Thr Ser Thr Ser Pro Thr Arg Ser195 200 205Met Ala Pro Gly Ala Val His Leu Pro Gln Pro Val Ser Thr Arg Ser210 215 220Gln Leu Asp Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro225 230 235 240Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 245 250 255Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 260 265 270Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe275 280 285Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro290 295 300Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr305 310 315 320Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 325 330 335Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 340 345 350Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg355 360 365Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly370 375 380Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro385 390 395 400Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 405 410 415Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 420 425 430Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His435 440 445Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys450 455 460135384DNAArtificialrecombinant plasmid p-puro 13agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc 60acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc 120tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 180ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccagattta 240attaaggctg cgcgctcgct cgctcactga ggccgcccgg gcaaagcccg ggcgtcgggc 300gacctttggt cgcccggcct cagtgagcga gcgagcgcgc agagagggag tggccaactc 360catcactagg ggttccttgt agttaatgat taacccgcca tgctacttat ctacgtagcc 420atgctctagg aagatcggaa ttcgccctta agctagctag ttattaatag taatcaatta 480cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg 540gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc 600ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa 660ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca 720atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta 780cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt 840acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg 900acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca 960actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca 1020gagctggttt agtgaaccgt cagatcctgc agaagttggt cgtgaggcac tgggcaggta 1080agtatcaagg ttacaagaca ggtttaagga gaccaataga aactgggctt gtcgagacag 1140agaagactct tgcgtttctg ataggcacct attggtctta ctgacatcca ctttgccttt 1200ctctccacag gtgtccaggc ggccgcc atg acc gag tac aag ccc acg gtg cgc 1254 Met Thr Glu Tyr Lys Pro Thr Val Arg 1 5ctc gcc acc cgc gac gac gtc ccc cgg gcc gta cgc acc ctc gcc gcc 1302Leu Ala Thr Arg Asp Asp Val Pro Arg Ala Val Arg Thr Leu Ala Ala10 15 20 25gcg ttc gcc gac tac ccc gcc acg cgc cac acc gtc gac ccg gac cgc 1350Ala Phe Ala Asp Tyr Pro Ala Thr Arg His Thr Val Asp Pro Asp Arg 30 35 40cac atc gag cgg gtc acc gag ctg caa gaa ctc ttc ctc acg cgc gtc 1398His Ile Glu Arg Val Thr Glu Leu Gln Glu Leu Phe Leu Thr Arg Val 45 50 55ggg ctc gac atc ggc aag gtg tgg gtc gcg gac gac ggc gcc gcg gtg 1446Gly Leu Asp Ile Gly Lys Val Trp Val Ala Asp Asp Gly Ala Ala Val60 65 70gcg gtc tgg acc acg ccg gag agc gtc gaa gcg ggg gcg gtg ttc gcc 1494Ala Val Trp Thr Thr Pro Glu Ser Val Glu Ala Gly Ala Val Phe Ala75 80 85gag atc ggc ccg cgc atg gcc gag ttg agc ggt tcc cgg ctg gcc gcg 1542Glu Ile Gly Pro Arg Met Ala Glu Leu Ser Gly Ser Arg Leu Ala Ala90 95 100 105cag caa cag atg gaa ggc ctc ctg gcg ccg cac cgg ccc aag gag ccc 1590Gln Gln Gln Met Glu Gly Leu Leu Ala Pro His Arg Pro Lys Glu Pro 110 115 120gcg tgg ttc ctg gcc acc gtc ggc gtc tcg ccc gac cac cag ggc aag 1638Ala Trp Phe Leu Ala Thr Val Gly Val Ser Pro Asp His Gln Gly Lys 125 130 135ggt ctg ggc agc gcc gtc gtg ctc ccc gga gtg gag gcg gcc gag cgc 1686Gly Leu Gly Ser Ala Val Val Leu Pro Gly Val Glu Ala Ala Glu Arg140 145 150gcc ggg gtg ccc gcc ttc ctg gag acc tcc gcg ccc cgc aac ctc ccc 1734Ala Gly Val Pro Ala Phe Leu Glu Thr Ser Ala Pro Arg Asn Leu Pro155 160 165ttc tac gag cgg ctc ggc ttc acc gtc acc gcc gac gtc gag gtg ccc 1782Phe Tyr Glu Arg Leu Gly Phe Thr Val Thr Ala Asp Val Glu Val Pro170 175 180 185gaa gga ccg cgc acc tgg tgc atg acc cgc aag ccc ggt gcc tga 1827Glu Gly Pro Arg Thr Trp Cys Met Thr Arg Lys Pro Gly Ala 190 195taagcttgga tccaatcaac ctctggatta caaaatttgt gaaagattga ctggtattct 1887taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc 1947tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt tgctgtctct 2007ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga 2067cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg ggactttcgc 2127tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc gctgctggac 2187aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaagc tgacgtcctt 2247tccatggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct tctgctacgt 2307cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc 2367tcttccgcgt cttcgagatc tgcctcgact gtgccttcta gttgccagcc atctgttgtt 2427tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa 2487taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg 2547gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggactcg 2607agttaagggc gaattcccga ttaggatctt cctagagcat ggctacgtag ataagtagca 2667tggcgggtta atcattaact acaaggaacc cctagtgatg gagttggcca ctccctctct 2727gcgcgctcgc tcgctcactg aggccgggcg accaaaggtc gcccgacgcc cgggctttgc 2787ccgggcggcc tcagtgagcg agcgagcgcg cagccttaat taacctaatt cactggccgt 2847cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc 2907acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca 2967acagttgcgc agcctgaatg gcgaatggga cgcgccctgt agcggcgcat taagcgcggc 3027gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc 3087tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa 3147tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact 3207tgattagggt gatggttcac gtagtgggcc atcgccccga tagacggttt ttcgcccttt 3267gacgctggag ttcacgttcc tcaatagtgg actcttgttc caaactggaa caacactcaa 3327ccctatctcg gtctattctt ttgatttata agggattttt ccgatttcgg cctattggtt 3387aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttat 3447aatttcaggt ggcatctttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta 3507aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc

ttcaataata 3567ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc 3627ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga 3687agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaatagtg gtaagatcct 3747tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg 3807tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta 3867ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat 3927gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt 3987acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga 4047tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga 4107gcgtgacacc acgatgcctg tagtaatggt aacaacgttg cgcaaactat taactggcga 4167actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc 4227aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc 4287cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg 4347tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat 4407cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata 4467tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct 4527ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga 4587ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg 4647cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc 4707aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct 4767agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc 4827tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt 4887ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg 4947cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct 5007atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag 5067ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag 5127tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg 5187gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg 5247cggttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac 5307cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt 5367gagcgaggaa gcggaag 538414199PRTArtificialSynthetic Construct 14Met Thr Glu Tyr Lys Pro Thr Val Arg Leu Ala Thr Arg Asp Asp Val1 5 10 15Pro Arg Ala Val Arg Thr Leu Ala Ala Ala Phe Ala Asp Tyr Pro Ala 20 25 30Thr Arg His Thr Val Asp Pro Asp Arg His Ile Glu Arg Val Thr Glu35 40 45Leu Gln Glu Leu Phe Leu Thr Arg Val Gly Leu Asp Ile Gly Lys Val50 55 60Trp Val Ala Asp Asp Gly Ala Ala Val Ala Val Trp Thr Thr Pro Glu65 70 75 80Ser Val Glu Ala Gly Ala Val Phe Ala Glu Ile Gly Pro Arg Met Ala 85 90 95Glu Leu Ser Gly Ser Arg Leu Ala Ala Gln Gln Gln Met Glu Gly Leu 100 105 110Leu Ala Pro His Arg Pro Lys Glu Pro Ala Trp Phe Leu Ala Thr Val115 120 125Gly Val Ser Pro Asp His Gln Gly Lys Gly Leu Gly Ser Ala Val Val130 135 140Leu Pro Gly Val Glu Ala Ala Glu Arg Ala Gly Val Pro Ala Phe Leu145 150 155 160Glu Thr Ser Ala Pro Arg Asn Leu Pro Phe Tyr Glu Arg Leu Gly Phe 165 170 175Thr Val Thr Ala Asp Val Glu Val Pro Glu Gly Pro Arg Thr Trp Cys 180 185 190Met Thr Arg Lys Pro Gly Ala195